Stem cell culture media and methods of enhancing cell survival

ABSTRACT

The invention provides improved methods for preparing hematopoietic cells for transplantation and the resulting improved hematopoietic cell compositions. The invention further relates to improved culture media and methods of culturing, processing, modulating, and expanding blood cell products for hematopoietic transplantation.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/209,131, filed Mar. 13, 2014, which claims the benefit under 35U.S.C. § 119(e) of U.S. Provisional Application No. 61/792,818, filedMar. 15, 2013, which is incorporated by reference in its entirety.

BACKGROUND Technical Field

The invention relates generally to improved culture media formanipulating cell populations, particularly populations of cellscomprising hematopoietic stem cells, compositions thereof, and methodsof using such media and compositions.

Description of the Related Art

Stem cells have utility for many clinical and research applications. Forexample, stem cells and their differentiated progeny can be used incellular assays, drug screening, and toxicity assays. Stem cells alsoshow promise for cell-based therapies, such as in regenerative medicinefor the treatment of damaged tissue. One aspect of regenerative medicinebeing pursued is the use of hematopoietic stem cell transplants to treatan expanding list of cancers and degenerative disorders. According tothe National Marrow Donor Program® (NMDP), an estimated 45,000 to 50,000hematopoietic cell transplants (bone marrow, peripheral blood stem cells(PBSC), or cord blood transplants) are performed annually worldwide totreat patients with life-threatening malignant and non-malignantdiseases (Horowitz M M. Uses and Growth of Hematopoietic CellTransplantation. In: Blume K G, Forman S J, Appelbaum F R, eds. Thomas'Hematopoietic Cell Transplantation. 3rd ed. Malden, Mass: Blackwell;2004:9-15). Moreover, approximately 4,800 patients are transplantedannually using unrelated donors or cord blood units through the NMDP.

For all of these applications, reproducible stem cell culture methodsare needed to provide adequate numbers of cells of suitable quality forthe purpose for which they are to be used. However, the art currentlylacks any cell culture media that can preserve the biological activitiesof cells that undergo cell processing activities, including suchprocesses as cryopreservation, thawing, resuspension, expansion,culturing, and maintenance of cell populations result in some degree ofcell death. Thus, the art is in need of efficient cell culture media forprocessing, maintaining, manipulating, and expanding populations ofcells for research and therapeutic purposes.

BRIEF SUMMARY

The invention generally provides improved culture media for manipulatingcell populations, compositions thereof, and methods of using such mediaand compositions.

In various embodiments, a culture medium is provided comprising: (a)about 1% to about 20% polysaccharide; and (b) a chemically defined cellculture medium.

In a particular embodiment, the polysaccharide is a dextran.

In one embodiment, the polysaccharide is a dextran selected from thegroup consisting of: dextran-1, dextran-10, dextran-20, dextran-30, anddextran-40.

In a certain embodiment, the polysaccharide is dextran-40.

In an additional embodiment, the polysaccharide is a hydroxyethyl starch(HES).

In a certain embodiment, the polysaccharide is a HES selected from thegroup consisting of: hetastarch, hexastarch, pentastarch, andtetrastarch.

In one embodiment, the polysaccharide is hetastarch.

In another embodiment, the medium comprises about 1% to about 5%polysaccharide.

In yet another embodiment, the medium comprises about 6% polysaccharide.

In one particular embodiment, the medium comprises about 7%polysaccharide.

In one certain embodiment, the medium comprises about 8% polysaccharide.

In one additional embodiment, the medium comprises about 9%polysaccharide.

In one further embodiment, the medium comprises about 10%polysaccharide.

In a particular embodiment, the medium comprises about 1% to about 5%HSA.

In a further embodiment, the medium comprises about 2% HSA.

In another embodiment, the medium comprises about 3% HSA.

In an additional embodiment, the medium comprises about 4% HSA.

In one embodiment, the medium comprises about 5% HSA.

In a further embodiment, wherein the chemically defined cell culturemedium is selected from the group consisting of: STEMSPAN-ACF,STEMSPAN-H3000, STEMSPAN-SFEM, STEMLINE II, STEMPRO 34, STEMSVIVO,Iscove's modified Dulbecco's medium (IMDM), Dulbecco's modified Eaglemedium (DMEM), Roswell Park Memorial Institute medium (RPMI) 1640medium, McCoy's 5A medium, minimum essential medium alpha medium(alpha-MEM), basal medium Eagle (BME), Fischer's medium, medium199,F-12K nutrient mixture medium (Kaighn's modification, F-12K), and X-VIVO20.

In a particular embodiment, the chemically defined cell culture mediumis selected from the group consisting of: STEMSPAN-ACF, STEMSPAN-H3000,and STEMSPAN-SFEM.

In a certain embodiment, the chemically defined cell culture medium isSTEMSPAN.

In another certain embodiment, the culture medium further comprises oneor more growth factors or cytokines.

In one certain embodiment, the chemically defined culture mediumcomprises one or more growth factors or cytokines selected from thegroup consisting of: flt3-ligand (FLT3); thrombopoietin (TPO), stem cellfactor (SCF), epidermal growth factor (EGF), transforming growthfactor-beta (TGF-β), basic fibroblast growth factor (bFGF),interleukin-3 (IL3), interleukin-6 (IL6), and interleukin-9 (IL9).

In one embodiment, the culture medium further comprises an agentselected from the group consisting of a cAMP analogue or enhancer, aGα-s activator, and a prostaglandin pathway agonist.

In a particular embodiment, the prostaglandin pathway agonistselectively binds the PGE2 EP2 or PGE2 EP4 receptor.

In an additional embodiment, the prostaglandin pathway agonist comprisesPGE2, or a PGE2 analogue or derivative.

In a further embodiment, the prostaglandin pathway agonist is selectedfrom the group consisting of: PGE2, 16,16-dmPGE2, 15(S)-15-methyl PGE2,20-ethyl PGE2, and 8-iso-16-cyclohexyl-tetranor PGE2.

In a further particular embodiment, the prostaglandin pathway agonistcomprises 16,16-dmPGE2.

In various embodiments, a composition is provided comprising: (a) apopulation of cells comprising hematopoietic cells; (b) about 1% toabout 20% polysaccharide; and (c) a chemically defined cell culturemedium.

In one embodiment, the population of cells is selected from the groupconsisting of: bone marrow cells (BMCs), umbilical cord blood cells(UCBCs), placental blood cells, mobilized peripheral blood cells(mPBCs), hematopoietic stem cells (HSCs), hematopoietic progenitor cells(HPCs), and CD34+ cells.

In a particular embodiment, the population of cells is selected from thegroup consisting of: bone marrow, umbilical cord blood, placental blood,or mobilized peripheral blood.

In another particular embodiment, the hematopoietic cells are selectedfrom the group consisting of: hematopoietic stem cells (HSCs),hematopoietic progenitor cells (HPCs), and CD34+ cells.

In a certain embodiment, the hematopoietic cells comprise a purifiedpopulation of CD34+ cells.

In an additional embodiment, the polysaccharide is a dextran.

In another embodiment, the polysaccharide is a dextran selected from thegroup consisting of: dextran-1, dextran-10, dextran-20, dextran-30, anddextran-40.

In one embodiment, the polysaccharide is dextran-40.

In a certain embodiment, the polysaccharide is a HES.

In a further embodiment, the polysaccharide is a HES selected from thegroup consisting of: hetastarch, hexastarch, pentastarch, andtetrastarch.

In a particular embodiment, the polysaccharide is hetastarch.

In an additional embodiment, the composition comprises about 1% to about5% polysaccharide.

In another embodiment, the composition comprises about 6%polysaccharide.

In a further certain embodiment, the composition comprises about 7%polysaccharide.

In one embodiment, the composition comprises about 8% polysaccharide.

In a particular embodiment, the composition comprises about 9%polysaccharide.

In a certain particular embodiment, the composition comprises about 10%polysaccharide.

In an additional particular embodiment, the composition comprises about1% to about 5% HSA.

In a further particular embodiment, the composition comprises about 2%HSA.

In another particular embodiment, the composition comprises about 3%HSA.

In one particular embodiment, the composition comprises about 4% HSA.

In one embodiment, the composition comprises about 5% HSA.

In an additional embodiment, the chemically defined cell culture mediumis selected from the group consisting of: STEMSPAN-ACF, STEMSPAN-H3000,STEMSPAN-SFEM, STEMLINE II, STEMPRO 34, STEMSVIVO, Iscove's modifiedDulbecco's medium (IMDM), Dulbecco's modified Eagle medium (DMEM),Roswell Park Memorial Institute medium (RPMI) 1640 medium, McCoy's 5Amedium, minimum essential medium alpha medium (alpha-MEM), basal mediumEagle (BME), Fischer's medium, medium199, F-12K nutrient mixture medium(Kaighn's modification, F-12K), and X-vivo 20.

In one embodiment, the chemically defined cell culture medium isselected from the group consisting of: STEMSPAN-ACF, STEMSPAN-H3000, andSTEMSPAN-SFEM.

In another embodiment, the chemically defined cell culture medium isSTEMSPAN.

In yet another embodiment, the composition further comprises one or moregrowth factors or cytokines.

In a particular embodiment, the chemically defined culture mediumcomprises one or more growth factors or cytokines selected from thegroup consisting of: flt3-ligand (FLT3); thrombopoietin (TPO), stem cellfactor (SCF), interleukin-3 (IL3), interleukin-6 (IL6), andinterleukin-9 (IL9).

In a certain embodiment, the composition further comprises an agentselected from the group consisting of: a cAMP analogue or enhancer, aGα-s activator, and a prostaglandin pathway agonist.

In a further embodiment, the prostaglandin pathway agonist selectivelybinds the PGE2 EP2 or PGE2 EP4 receptor.

In a particular embodiment, the prostaglandin pathway agonist comprisesPGE2, or a PGE2 analogue or derivative.

In an additional embodiment, the prostaglandin pathway agonist isselected from the group consisting of: PGE2, 16,16-dmPGE2,15(S)-15-methyl PGE2, 20-ethyl PGE2, and 8-iso-16-cyclohexyl-tetranorPGE2.

In one embodiment, the prostaglandin pathway agonist comprises16,16-dmPGE2.

In various embodiments, a method is provided for stabilizing ahematopoietic cell population for transplantation comprising: (a)thawing a cryopreserved hematopoietic cell population; and (b)transferring the thawed hematopoietic cell population into any one ofthe culture media according to any of the foregoing embodiments; whereinthe transferred hematopoietic cell population has reduced cell lysis andincreased CD34+ cell viability compared to a thawed controlhematopoietic cell population that has been transferred to a controlsolution.

In a particular embodiment, the hematopoietic cell population is thawedat a temperature of about 20° C. to about 37° C.

In another embodiment, the hematopoietic cell population is thawed at atemperature of about 25° C.

In a further embodiment, the hematopoietic cell population is thawed ata temperature of about 30° C.

In one embodiment, the hematopoietic cell population is thawed at atemperature of about 37° C.

In a particular embodiment, the hematopoietic cell population isselected from the group consisting of: bone marrow cells (BMCs),umbilical cord blood cells (UCBCs), placental blood cells, mobilizedperipheral blood cells (mPBCs), hematopoietic stem cells (HSCs),hematopoietic progenitor cells (HPCs), and CD34⁺ cells.

In an additional embodiment, the hematopoietic cell population isselected from the group consisting of: bone marrow, umbilical cordblood, placental blood, or mobilized peripheral blood.

In another embodiment, the hematopoietic cell population is selectedfrom the group consisting of: hematopoietic stem cells (HSCs),hematopoietic progenitor cells (HPCs), and CD34⁺ cells.

In a certain embodiment, the hematopoietic cell population is a purifiedpopulation of CD34⁺ cells.

In another embodiment, the cell lysis of the hematopoietic cellpopulation is decreased about 10% compared to the cell lysis of thecontrol hematopoietic cell population.

In one embodiment, the cell lysis of the hematopoietic cell populationis decreased about 20% compared to the cell lysis of the controlhematopoietic cell population.

In another embodiment, the cell lysis of the hematopoietic cellpopulation is decreased about 30% compared to the cell lysis of thecontrol hematopoietic cell population.

In an additional embodiment, the cell lysis of the hematopoietic cellpopulation is decreased about 40% compared to the cell lysis of thecontrol hematopoietic cell population.

In a certain embodiment, the cell lysis of the hematopoietic cellpopulation is decreased about 50% compared to the cell lysis of thecontrol hematopoietic cell population.

In a further embodiment, the cell lysis of the hematopoietic cellpopulation is decreased about two-fold compared to the cell lysis of thecontrol hematopoietic cell population.

In a particular embodiment, the cell lysis of the hematopoietic cellpopulation is decreased about three-fold compared to the cell lysis ofthe control hematopoietic cell population.

In a further embodiment, the cell lysis of the hematopoietic cellpopulation is decreased about five-fold compared to the cell lysis ofthe control hematopoietic cell population.

In one embodiment, the CD34+ cell viability of the hematopoietic cellpopulation is increased about 10% compared to the CD34+ cell viabilityof the control hematopoietic cell population.

In an additional embodiment, the CD34+ cell viability of thehematopoietic cell population is increased about 20% compared to theCD34+ cell viability of the control hematopoietic cell population.

In a particular embodiment, the CD34+ cell viability of thehematopoietic cell population is increased about 30% compared to theCD34+ cell viability of the control hematopoietic cell population.

In another particular embodiment, the CD34+ cell viability of thehematopoietic cell population is increased about 40% compared to theCD34+ cell viability of the control hematopoietic cell population.

In another embodiment, the CD34+ cell viability of the hematopoieticcell population is increased about 50% compared to the CD34+ cellviability of the control hematopoietic cell population.

In one embodiment, the CD34+ cell viability of the hematopoietic cellpopulation is increased about two-fold compared to the CD34+ cellviability of the control hematopoietic cell population.

In one embodiment, the CD34+ cell viability of the hematopoietic cellpopulation is increased about three-fold compared to the CD34+ cellviability of the control hematopoietic cell population.

In a particular embodiment, the CD34+ cell viability of thehematopoietic cell population is increased about five-fold compared tothe CD34+ cell viability of the control hematopoietic cell population.

In a certain particular embodiment, the hematopoietic cell population ismodulated ex VIVO.

In an additional embodiment, the modulation comprises contacting thehematopoietic cell population with an agent selected from the groupconsisting of: a cAMP analogue or enhancer, a Gα-s activator, and aprostaglandin pathway agonist.

In a certain embodiment, the prostaglandin pathway agonist selectivelybinds the PGE2 EP2 or PGE2 EP4 receptor.

In an additional certain embodiment, the prostaglandin pathway agonistcomprises PGE2, or a PGE2 analogue or derivative.

In a certain further embodiment, the prostaglandin pathway agonist isselected from the group consisting of: PGE2, 16,16-dmPGE2,15(S)-15-methyl PGE2, 20-ethyl PGE2, and 8-iso-16-cyclohexyl-tetranorPGE2.

In one embodiment, the prostaglandin pathway agonist comprises16,16-dmPGE2.

In a further embodiment, the hematopoietic cell population is contactedwith the at least one agent for a time of about one hour to about fourhours.

In a particular embodiment, the hematopoietic cell population iscontacted with the at least one agent for a time of about one hour.

In an additional embodiment, the hematopoietic cell population iscontacted with the at least one agent for a time of about two hours.

In a certain embodiment, the hematopoietic cell population is contactedwith the at least one agent at a temperature of about 25° C. to about37° C.

In another embodiment, the hematopoietic cell population is contactedwith the at least one agent at a temperature of about 30° C.

In one embodiment, the hematopoietic cell population is contacted withthe at least one agent at a temperature of about 37° C.

In a particular embodiment, the hematopoietic cell population iscontacted with 10 μM 16,16-dmPGE2, at about 37° C., for about two hours.

In an additional embodiment, engraftment of the hematopoietic cellpopulation is increased in vivo, compared to a non-modulatedhematopoietic cell population.

In another additional embodiment, reconstitution of the hematopoieticcell population is increased in vivo, compared to a non-modulatedhematopoietic cell population.

In an additional embodiment, homing of the hematopoietic cell populationis increased in vivo, compared to a non-modulated hematopoietic cellpopulation.

In one embodiment, proliferation of the hematopoietic cell population isincreased in vivo, compared to a non-modulated hematopoietic cellpopulation.

In one embodiment, expression of at least two genes selected from thegroup consisting of: CREM, GEM, NR4A2, NR4A3, ILIA, COX2, HEY1, CXCL2,CXCL3, and ULBP2 is increased by about 20-fold in the hematopoietic cellpopulation compared to expression of the at least two genes in thecontrol hematopoietic cell population.

In one particular embodiment, expression of at least five genes selectedfrom the group consisting of: CREM, GEM, NR4A2, NR4A3, ILIA, COX2, HEYI,CXCL2, CXCL3, and ULBP2 is increased by about 10-fold in thehematopoietic cell population compared to expression of the at leastfive genes in the control hematopoietic cell population.

In another particular embodiment, expression of at least five genesselected from the group consisting of: CREM, GEM, NR4A2, NR4A3, ILIA,COX2, HEYI, CXCL2, CXCL3, and ULBP2 is increased by about 3-fold in thehematopoietic cell population compared to expression of the at leastfive genes in the control hematopoietic cell population.

In a certain particular embodiment, expression of at least five genesselected from the group consisting of: CREM, GEM, NR4A2, NR4A3, ILIA,COX2, HEYI, CXCL2, CXCL3, and ULBP2 is increased by about 2-fold in thehematopoietic cell population compared to expression of the at leastfive genes in the control hematopoietic cell population.

In a further particular embodiment, expression of the genes CREM, GEM,NR4A2, NR4A3, ILIA, COX2, HASI , CXCL2, CXCL3, and CXCR4 is eachincreased by about 3-fold in the hematopoietic cell population comparedto expression of the genes in the control hematopoietic cell population.

In an additional particular embodiment, expression of the genes CREM,GEM, NR4A2, NR4A3, ILIA, COX2, HASI, CXCL2, CXCL3, and CXCR4 is eachincreased by about 2-fold in the hematopoietic cell population comparedto expression of the genes in the control hematopoietic cell population.

In one embodiment, the hematopoietic cell population is administered toa subject.

In another embodiment, the hematopoietic cell population is allogeneicto the subject.

In yet another embodiment, the hematopoietic cell population isautologous to the subject.

In one particular embodiment, the subject has a disease, disorder, orcondition selected from the group consisting of: ischemia, a nonmalignant blood disorder, an immunodeficiency, severe combinedimmunodeficiency (SCID), lymphocytopenia, thrombocytopenia, neutropenia,anemia, Fanconi's anemia, severe aplastic anemia, a congenitalhemoglobinopathy, sickle cell disease, -thalassemaia, sickle-celldisease, Wiskott-Aldrich syndrome, a metabolic storage disease, Hurler'sdisease, Hunter's disease, mannosidosis, a cancer, a hematologicalmalignancy, acute leukemia, chronic myeloid leukemia chronic lymphoidleukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma,myelodysplastic syndrome, a non-hematological cancer, breast cancer,ovarian cancer, brain cancer, prostate cancer, lung cancer, coloncancer, skin cancer, liver cancer, and pancreatic cancer.

In various embodiments, a method is provided for increasing viability ofa hematopoietic cell population for transplantation comprising: (a)thawing a cryopreserved hematopoietic cell population; and (b)transferring the thawed hematopoietic cell population into any one ofthe culture media according to any one of the foregoing embodiments.

In one embodiment, the hematopoietic cell population is thawed at atemperature of about 20° C. to about 37° C.

In a particular embodiment, the hematopoietic cell population is thawedat a temperature of about 25° C.

In a further embodiment, the hematopoietic cell population is thawed ata temperature of about 30° C.

In a further embodiment, the hematopoietic cell population is thawed ata temperature of about 37° C.

In an additional embodiment, the hematopoietic cell population isselected from the group consisting of: bone marrow cells (BMCs),umbilical cord blood cells (UCBCs), placental blood cells, mobilizedperipheral blood cells (mPBCs), hematopoietic stem cells (HSCs),hematopoietic progenitor cells (HPCs), and CD34+ cells.

In one embodiment, the hematopoietic cell population is selected fromthe group consisting of: bone marrow, umbilical cord blood, placentalblood, or mobilized peripheral blood.

In a particular embodiment, the hematopoietic cell population isselected from the group consisting of: hematopoietic stem cells (HSCs),hematopoietic progenitor cells (HPCs), and CD34+ cells.

In a certain embodiment, the hematopoietic cell population is a purifiedpopulation of CD34+ cells.

In another embodiment, the cell lysis of the hematopoietic cellpopulation is decreased about 10% compared to the cell lysis of a thawedcontrol hematopoietic cell population that has been transferred to acontrol solution.

In a further embodiment, the cell lysis of the hematopoietic cellpopulation is decreased about 20% compared to the cell lysis of a thawedcontrol hematopoietic cell population that has been transferred to acontrol solution.

In one embodiment, the cell lysis of the hematopoietic cell populationis decreased about 30% compared to the cell lysis of a thawed controlhematopoietic cell population that has been transferred to a controlsolution.

In an additional embodiment, the cell lysis of the hematopoietic cellpopulation is decreased about 40% compared to the cell lysis of a thawedcontrol hematopoietic cell population that has been transferred to acontrol solution.

In a particular embodiment, the cell lysis of the hematopoietic cellpopulation is decreased about 50% compared to the cell lysis of a thawedcontrol hematopoietic cell population that has been transferred to acontrol solution.

In a particular embodiment, the cell lysis of the hematopoietic cellpopulation is decreased about two-fold compared to the cell lysis of athawed control hematopoietic cell population that has been transferredto a control solution.

In one certain embodiment, the cell lysis of the hematopoietic cellpopulation is decreased about three-fold compared to the cell lysis of athawed control hematopoietic cell population that has been transferredto a control solution.

In a further embodiment, the cell lysis of the hematopoietic cellpopulation is decreased about five-fold compared to the cell lysis of athawed control hematopoietic cell population that has been transferredto a control solution.

In one embodiment, the CD34+ cell viability of the hematopoietic cellpopulation is increased about 10% compared to the CD34+ cell viabilityof a thawed control hematopoietic cell population that has beentransferred to a control solution.

In one embodiment, the CD34+ cell viability of the hematopoietic cellpopulation is increased about 20% compared to the CD34+ cell viabilityof a thawed control hematopoietic cell population that has beentransferred to a control solution.

In a particular embodiment, the CD34+ cell viability of thehematopoietic cell population is increased about 30% compared to theCD34+ cell viability of a thawed control hematopoietic cell populationthat has been transferred to a control solution.

In another embodiment, the CD34+ cell viability of the hematopoieticcell population is increased about 40% compared to the CD34+ cellviability of a thawed control hematopoietic cell population that hasbeen transferred to a control solution.

In an additional embodiment, the CD34+ cell viability of thehematopoietic cell population is increased about 50% compared to theCD34+ cell viability of a thawed control hematopoietic cell populationthat has been transferred to a control solution.

In a further embodiment, the CD34+ cell viability of the hematopoieticcell population is increased about two-fold compared to the CD34+ cellviability of a thawed control hematopoietic cell population that hasbeen transferred to a control solution.

In a further particular embodiment, the CD34+ cell viability of thehematopoietic cell population is increased about three-fold compared tothe CD34+ cell viability of a thawed control hematopoietic cellpopulation that has been transferred to a control solution.

In a particular embodiment, the CD34+ cell viability of thehematopoietic cell population is increased about five-fold compared tothe CD34+ cell viability of a thawed control hematopoietic cellpopulation that has been transferred to a control solution.

In various embodiments, a method is provided for increasing totalnucleated cell (TNC) count of a cryopreserved blood cell product fortransplantation comprising: (a) thawing a cryopreserved blood cellproduct; and (b) transferring the thawed blood cell product into any oneof the culture media according to any one of the foregoing embodiments.

In a certain embodiment,n the blood cell product is thawed at atemperature of about 20° C. to about 37° C.

In another certain embodiment, the blood cell product is thawed at atemperature of about 25° C.

In one embodiment, the blood cell product is thawed at a temperature ofabout 30° C.

In a particular embodiment, the blood cell product is thawed at atemperature of about 37° C.

In an additional embodiment, the blood cell product is selected from thegroup consisting of: bone marrow cells (BMCs), umbilical cord bloodcells (UCBCs), placental blood cells, mobilized peripheral blood cells(mPBCs), hematopoietic stem cells (HSCs), hematopoietic progenitor cells(HPCs), and CD34+ cells.

In one embodiment, the blood cell product is selected from the groupconsisting of bone marrow, umbilical cord blood, placental blood, ormobilized peripheral blood.

In one particular embodiment, the blood cell product is selected fromthe group consisting of: hematopoietic stem cells (HSCs), hematopoieticprogenitor cells (HPCs), and CD34+ cells.

In another embodiment, the blood cell product is a purified populationof CD34+ cells.

In a further embodiment, the TNC count of the blood cell product isincreased about 10% compared to the TNC count of a thawed control bloodcell product that has been transferred to a control solution.

In a particular embodiment, the TNC count of the blood cell product isincreased about 20% compared to the TNC count of a thawed control bloodcell product that has been transferred to a control solution.

In a further particular embodiment, the TNC count of the blood cellproduct is increased about 30% compared to the TNC count of a thawedcontrol blood cell product that has been transferred to a controlsolution.

In one embodiment, the TNC count of the blood cell product is increasedabout 40% compared to the TNC count of a thawed control blood cellproduct that has been transferred to a control solution.

In a certain embodiment, the TNC count of the blood cell product isincreased about 50% compared to the TNC count of a thawed control bloodcell product that has been transferred to a control solution.

In a particular embodiment, the TNC count of the blood cell product isincreased about two-fold compared to the TNC count of a thawed controlblood cell product that has been transferred to a control solution.

In an additional embodiment, the TNC count of the blood cell product isincreased about three-fold compared to the TNC count of a thawed controlblood cell product that has been transferred to a control solution.

In an additional embodiment, the TNC count of the blood cell product isincreased about five-fold compared to the TNC count of a thawed controlblood cell product that has been transferred to a control solution.

In various embodiments, a method is provided for preparing cryopreservedblood cell products for transplantation comprising: (a) thawing acryopreserved blood cell product; and (b) transferring the thawed bloodcell product into any one of the culture media according to any one ofthe foregoing embodiments.

In another embodiment, the blood cell product is thawed at a temperatureof about 20° C. to about 37° C.

In a further embodiment, the blood cell product is thawed at atemperature of about 25° C.

In a particular embodiment, the blood cell product is thawed at atemperature of about 30° C.

In one embodiment, the blood cell product is thawed at a temperature ofabout 37° C.

In one additional embodiment, the blood cell product is selected fromthe group consisting of: bone marrow cells (BMCs), umbilical cord bloodcells (UCBCs), placental blood cells, mobilized peripheral blood cells(mPBCs), hematopoietic stem cells (HSCs), hematopoietic progenitor cells(HPCs), and CD34+ cells.

In a particular embodiment, the blood cell product is selected from thegroup consisting of: bone marrow, umbilical cord blood, placental blood,or mobilized peripheral blood.

In a certain embodiment, the blood cell product is selected from thegroup consisting of: hematopoietic stem cells (HSCs), hematopoieticprogenitor cells (HPCs), and CD34+ cells.

In one embodiment, the blood cell product is a purified population ofCD34+ cells.

In one further embodiment, the blood cell product is modulated ex vivo.

In one certain embodiment, the modulation comprises contacting the bloodcell product with an agent selected from the group consisting of: a cAMPanalogue or enhancer, a Ga-s activator, and a prostaglandin pathwayagonist.

In one particular embodiment, the prostaglandin pathway agonistselectively binds the PGE2 EP2 or PGE2 EP4 receptor.

In one additional embodiment, the prostaglandin pathway agonistcomprises PGE2, or a PGE2 analogue or derivative.

In a particular embodiment, the prostaglandin pathway agonist isselected from the group consisting of: PGE2, 16,16-dmPGE2,15(S)-15-methyl PGE2, 20-ethyl PGE2, and 8-iso-16-cyclohexyl-tetranorPGE2.

In another particular embodiment, the prostaglandin pathway agonistcomprises 16,16-dmPGE2.

In one particular embodiment, the blood cell product is contacted withthe at least one agent for a time of about one hour to about four hours.

In a further particular embodiment, the blood cell product is contactedwith the at least one agent for a time of about one hour.

In a certain particular embodiment, the blood cell product is contactedwith the at least one agent for a time of about two hours.

In an additional particular embodiment, the blood cell product iscontacted with the at least one agent at a temperature of about 25° C.to about 37° C.

In an additional embodiment, the blood cell product is contacted withthe at least one agent at a temperature of about 30° C.

In a certain embodiment, the blood cell product is contacted with the atleast one agent at a temperature of about 37° C.

In one embodiment, the blood cell product is contacted with 10 μM16,16-dmPGE2, at about 37° C., for about two hours.

In another embodiment, engraftment of the blood cell product isincreased in vivo, compared to a non-modulated blood cell product.

In yet another embodiment, reconstitution of the blood cell product isincreased in vivo, compared to a non-modulated blood cell product.

In still yet another embodiment, homing of the blood cell product isincreased in vivo, compared to a non-modulated blood cell product.

In a particular embodiment, proliferation of the blood cell product isincreased in vivo, compared to a non-modulated blood cell product.

In an additional embodiment, expression of at least two genes selectedfrom the group consisting of: CREM, GEM, NR4A2, NR4A3, ILIA, COX2, HEYI,CXCL2, CXCL3, and ULBP2 is increased by about 20-fold in the blood cellproduct compared to expression of the at least two genes in the controlblood cell product.

In a further embodiment, expression of at least five genes selected fromthe group consisting of: CREM, GEM, NR4A2, NR4A3, ILIA, COX2, HEYI,CXCL2, CXCL3, and ULBP2 is increased by about 10-fold in the blood cellproduct compared to expression of the at least five genes in the controlblood cell product.

In one embodiment, expression of at least five genes selected from thegroup consisting of: CREM, GEM, NR4A2, NR4A3, ILIA, COX2, HEYI, CXCL2,CXCL3, and ULBP2 is increased by about 3-fold in the blood cell productcompared to expression of the at least five genes in the control bloodcell product.

In a particular embodiment, expression of at least five genes selectedfrom the group consisting of: CREM, GEM, NR4A2, NR4A3, ILIA, COX2, HEYI,CXCL2, CXCL3, and ULBP2 is increased by about 2-fold in the blood cellproduct compared to expression of the at least five genes in the controlblood cell product.

In a certain embodiment, expression of the genes CREM, GEM, NR4A2,NR4A3, ILIA, COX2, HASI , CXCL2, CXCL3, and CXCR4 is each increased byabout 3-fold in the blood cell product compared to expression of thegenes in the control blood cell product.

In another embodiment, expression of the genes CREM, GEM, NR4A2, NR4A3,ILIA, COX2, HASI, CXCL2, CXCL3, and CXCR4 is each increased by about2-fold in the blood cell product compared to expression of the genes inthe control blood cell product.

In one embodiment, the blood cell product is administered to a subject.

In a further embodiment, the blood cell product is allogeneic to thesubject.

In an additional embodiment, the blood cell product is autologous to thesubject.

In a particular embodiment, the subject has a disease, disorder, orcondition selected from the group consisting of: ischemia, a nonmalignant blood disorder, an immunodeficiency, severe combinedimmunodeficiency (SCID), lymphocytopenia, thrombocytopenia, neutropenia,anemia, Fanconi's anemia, severe aplastic anemia, a congenitalhemoglobinopathy, sickle cell disease, -thalassemaia, sickle-celldisease, Wiskott-Aldrich syndrome, a metabolic storage disease, Hurler'sdisease, Hunter's disease, mannosidosis, a cancer, a hematologicalmalignancy, acute leukemia, chronic myeloid leukemia chronic lymphoidleukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma,myelodysplastic syndrome, a non-hematological cancer, breast cancer,ovarian cancer, brain cancer, prostate cancer, lung cancer, coloncancer, skin cancer, liver cancer, and pancreatic cancer.

In various embodiments, a culture medium is provided according to anyone of the foregoing embodiments, wherein the culture medium maintains aTNC of at least 70% in a thawed whole cord blood sample modulated bycontacting the sample with an agent that modulates a prostaglandinpathway for a duration of about 1 to about 24 hours, at a temperature ofabout 25° C. to about 37° C.

In a particular embodiment, the culture medium maintains the TNC of atleast 75% in the thawed whole cord blood sample.

In one embodiment, the culture medium maintains the TNC of at least 80%in the thawed whole cord blood sample.

In a certain embodiment, the culture medium maintains the TNC of atleast 85% in the thawed whole cord blood sample.

In a certain particular embodiment, the culture medium maintains the TNCof at least 90% in the thawed whole cord blood sample.

In a certain additional embodiment, the culture medium maintains the TNCof at least 95% in the thawed whole cord blood sample.

In another certain embodiment, the culture medium maintains the TNC ofat least 99% in the thawed whole cord blood sample.

In a certain further embodiment, the agent is selected from the groupconsisting of: a cAMP analogue or enhancer, a Ga-s activator, and aprostaglandin pathway agonist.

In one particular embodiment, the agent selectively binds the PGE2 EP2or PGE2 EP4 receptor.

In a particular embodiment, the agent comprises PGE2, or a PGE2 analogueor derivative.

In one embodiment, the agent is selected from the group consisting of:PGE2, 16,16-dmPGE2, 15(S)-15-methyl PGE2, 20-ethyl PGE2, and8-iso-16-cyclohexyl-tetranor PGE2.

In a further embodiment, the agent comprises 16,16-dmPGE2.

In an additional embodiment, the whole cord blood sample is contactedwith the at least one agent for a time of about one hour to about fourhours.

In a certain embodiment, the whole cord blood sample is contacted withthe at least one agent for a time of about one hour.

In another embodiment, the whole cord blood sample is contacted with theat least one agent for a time of about two hours.

In one embodiment, the whole cord blood sample is contacted with the atleast one agent at a temperature of about 25° C. to about 37° C.

In one certain embodiment, the whole cord blood sample is contacted withthe at least one agent at a temperature of about 30° C.

In one particular embodiment, the whole cord blood sample is contactedwith the at least one agent at a temperature of about 37° C.

In another embodiment, the whole cord blood sample is contacted with 10μM 16,16-dmPGE2, at about 37° C., for about two hours.

In a particular embodiment, the contacted whole cord blood sampleexpresses at least two genes selected from the group consisting of:CREM, GEM, NR4A2, NR4A3, ILIA, COX2, HEYI, CXCL2, CXCL3, and ULBP2 about20-fold compared to expression of the at least two genes in a controlblood cell product.

In one particular embodiment, the contacted whole cord blood sampleexpresses at least five genes selected from the group consisting of:CREM, GEM, NR4A2, NR4A3, ILIA, COX2, HEYI, CXCL2, CXCL3, and ULBP2 about10-fold compared to expression of the at least five genes in a controlblood cell product.

In another particular embodiment, the contacted whole cord blood sampleexpresses at least five genes selected from the group consisting of:CREM, GEM, NR4A2, NR4A3, ILIA, COX2, HEYI, CXCL2, CXCL3, and ULBP2 about3-fold compared to expression of the at least five genes in a controlblood cell product.

In an additional particular embodiment, the contacted whole cord bloodsample expresses at least five genes selected from the group consistingof: CREM, GEM, NR4A2, NR4A3, ILIA, COX2, HEYI, CXCL2, CXCL3, and ULBP2about 2-fold compared to expression of the at least five genes in acontrol blood cell product.

In a further particular embodiment, the contacted whole cord bloodsample expresses the genes CREM, GEM, NR4A2, NR4A3, ILIA, COX2, HEYI,CXCL2, CXCL3, and ULBP2 about 10-fold compared to expression of thegenes in a control blood cell product.

In a certain particular embodiment, the contacted whole cord bloodsample expresses the genes CREM, GEM, NR4A2, NR4A3, ILIA, COX2, HEYI,CXCL2, CXCL3, and ULBP2 about 5-fold compared to expression of the genesin a control blood cell product.

In one embodiment, the contacted whole cord blood sample expresses thegenes CREM, GEM, NR4A2, NR4A3, ILIA, COX2, HEYI, CXCL2, CXCL3, and ULBP2about 3-fold compared to expression of the genes in a control blood cellproduct.

In a certain embodiment, the contacted whole cord blood sample expressesthe genes CREM, GEM, NR4A2, NR4A3, ILIA, COX2, HEYI, CXCL2, CXCL3, andULBP2 about 2-fold compared to expression of the genes in a controlblood cell product. In various embodiments, a whole cord blood samplethawed and transferred into a culture medium is provided according toany one of the foregoing embodiments, and modulated in the culturemedium by contacting the sample with an agent that modulates aprostaglandin pathway for a duration of about 1 to about 24 hours, at atemperature of about 25° C. to about 37° C., comprising a TNC of atleast 70%, wherein the sample is not subject to enrichment.

In a certain embodiment, the TNC is at least 75%. In a particularembodiment, the TNC is at least 80%. In one embodiment, the TNC is atleast 85%.

In an additional embodiment, the TNC is at least 90%. In anotherembodiment, the TNC is at least 95%.

In a particular embodiment, the TNC is at least 99%.

In a further embodiment, a thawed whole cord blood sample according toany one of the foregoing embodiments is provided, wherein the agent isselected from the group consisting of: a cAMP analogue or enhancer, aGa-s activator, and a prostaglandin pathway agonist.

In one embodiment, a thawed whole cord blood sample according to any oneof the foregoing embodiments is provided, wherein the agent selectivelybinds the PGE2 EP2 or PGE2 EP4 receptor.

In a particular embodiment, a thawed whole cord blood sample accordingto any one of the foregoing embodiments is provided, wherein the agentcomprises PGE2, or PGE2 analogue or derivative.

In a certain embodiment, a thawed whole cord blood sample according toany one of the foregoing embodiments is provided, wherein the agent isselected from the group consisting of: PGE2, 16,16-dmPGE2,15(S)-15-methyl PGE2, 20-ethyl PGE2, and 8-iso-16-cyclohexyl-tetranorPGE2.

In another embodiment, a thawed whole cord blood sample according to anyone of the foregoing embodiments is provided, wherein the agentcomprises 16,16-dmPGE2.

In an additional embodiment, a thawed whole cord blood sample accordingto any one of the foregoing embodiments is provided, wherein the thawedwhole cord blood sample is contacted with the at least one agent for atime of about one hour to about four hours.

In a further embodiment, a thawed whole cord blood sample according toany one of the foregoing embodiments is provided, wherein the thawedwhole cord blood sample is contacted with the at least one agent for atime of about one hour.

In a particular embodiment, a thawed whole cord blood sample accordingto any one of the foregoing embodiments, wherein the thawed whole cordblood sample is contacted with the at least one agent for a time ofabout two hours.

In another particular embodiment, a thawed whole cord blood sampleaccording to any one of the foregoing embodiments is provided, whereinthe thawed whole cord blood sample is contacted with the at least oneagent at a temperature of about 25° C. to about 37° C.

In one embodiment, a thawed whole cord blood sample according to any oneof the foregoing embodiments is provided, wherein the thawed whole cordblood sample is contacted with the at least one agent at a temperatureof about 30° C.

In another embodiment, a thawed whole cord blood sample according to anyone of the foregoing embodiments is provided, wherein the thawed wholecord blood sample is contacted with the at least one agent at atemperature of about 37° C.

In one embodiment, a thawed whole cord blood sample according to any oneof the foregoing embodiments is provided, wherein the thawed whole cordblood sample is contacted with 10 μM 16,16-dmPGE2, at about 37° C., forabout two hours.

In an additional embodiment, a thawed whole cord blood sample accordingto any one of the foregoing embodiments is provided, wherein the thawedwhole cord blood sample expresses at least two genes selected from thegroup consisting of: CREM, GEM, NR4A2, NR4A3, ILIA, COX2, HEYI, CXCL2,CXCL3, and ULBP2 about 20-fold compared to expression of the at leasttwo genes in a control blood cell product.

In a certain embodiment, a thawed whole cord blood sample according toany one of the foregoing embodiments is provided, wherein the thawedwhole cord blood sample expresses at least five genes selected from thegroup consisting of: CREM, GEM, NR4A2, NR4A3, ILIA, COX2, HEYI, CXCL2,CXCL3, and ULBP2 about 10-fold compared to expression of the at leastfive genes in a control blood cell product.

In an additional embodiment, a thawed whole cord blood sample accordingto any one of the foregoing embodiments is provided, wherein the thawedwhole cord blood sample expresses at least five genes selected from thegroup consisting of: CREM, GEM, NR4A2, NR4A3, ILIA, COX2, HEYI, CXCL2,CXCL3, and ULBP2 about 3-fold compared to expression of the at leastfive genes in a control blood cell product.

In one embodiment, a thawed whole cord blood sample according to any oneof the foregoing embodiments is provided, wherein the thawed whole cordblood sample expresses at least five genes selected from the groupconsisting of: CREM, GEM, NR4A2, NR4A3, ILIA, COX2, HEYI, CXCL2, CXCL3,and ULBP2 about 2-fold compared to expression of the at least five genesin a control blood cell product.

In a further embodiment, a thawed whole cord blood sample according toany one of the foregoing embodiments is provided, wherein the thawedwhole cord blood sample expresses the genes CREM, GEM, NR4A2, NR4A3,ILIA, COX2, HEYI, CXCL2, CXCL3, and ULBP2 about 10-fold compared toexpression of the genes in a control blood cell product.

In a certain embodiment, a thawed whole cord blood sample according toany one of the foregoing embodiments is provided, wherein the thawedwhole cord blood sample expresses the genes CREM, GEM, NR4A2, NR4A3,ILIA, COX2, HEYI, CXCL2, CXCL3, and ULBP2 about 5-fold compared toexpression of the genes in a control blood cell product.

In a particular embodiment, a thawed whole cord blood sample accordingto any one of the foregoing embodiments is provided,wherein the thawedwhole cord blood sample expresses the genes CREM, GEM, NR4A2, NR4A3,ILIA, COX2, HEYI, CXCL2, CXCL3, and ULBP2 about 3-fold compared toexpression of the genes in a control blood cell product.

In one embodiment, a thawed whole cord blood sample according to any oneof the foregoing embodiments is provided, wherein the thawed whole cordblood sample expresses the genes CREM, GEM, NR4A2, NR4A3, ILIA, COX2,HEYI, CXCL2, CXCL3, and ULBP2 about 2-fold compared to expression of thegenes in a control blood cell product.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A shows the number of viable CD34⁺ cells in the samples incubatedin STEMSPAN-ACF with 8% Dextran-40 compared to STEMSPAN-ACF alone atpost-thaw, post-wash, after one hour of incubation at 37° C. and aftertwo hours of incubation at 37° C. (post-incubation).

FIG. 1B shows the percentage of intact granulocytes in the CD45⁺ cellfraction in the sample incubated in STEMSPAN-ACF with 8% Dextrancompared to STEMSPAN alone at post-thaw, post-wash, after one hour ofincubation at 37° C. and after two hours of incubation at 37° C.(post-incubation).

FIG. 2 shows the TNC count for the cord blood units treated with 10 μMdmPGE₂ and processed in STEMSPAN-ACF, STEMSPAN-ACF+2.1% HSA,STEMSPAN+4.2% HSA, IMDM+4.2% HSA, or STEMSPAN-ACF with 8% Dextran-40 atpost-thaw, post-wash, and after two hours of incubation at 37° C.

FIG. 3 shows the percent of CD34⁺ cells that show increased CXCR4expression after incubation with 10 μM dmPGE2 for two hours at 37° C.Samples were treated with STEMSPAN-ACF, STEMSPAN-ACF+5% Dextran-40, orSTEMSPAN+10% Dextran-40.

DETAILED DESCRIPTION A. Overview

The invention provides improved cell culture media for processing andmodulating populations of cells, particularly compositions ofhematopoietic stem cells. The invention also provides methods ofpreparing hematopoietic cell compositions for transplant therapy.Without wishing to be bound to any particular theory, it is contemplatedthat the culture media improves cell viability and reduces cell lysis ofvarious cell types in the cell population (e.g., white blood cells,including granulocytes) during cell processing, including duringpreparation of the thawed cell populations, and during subsequent exvivo manipulations of the cells. The cell culture media may decreasecell lysis by stabilizing the membranes of apoptosing cells.

In addition to a resulting increase in total nucleated cell count (TNC)during cell processing as a result of increasing cell stability anddecreasing cell lysis (by preventing the release of intracellularcomponents), the cell culture media prevents cell debris aggregation inthe blood cell product that may cause “clumping” within the cellproduct. Clumping in blood cell products may hinder further cellprocessing manipulation of the cell, and may also inhibit cellularprocesses, including modulation of the cells. Thus, by inhibitingapoptosis and lysis of cells (e.g., white blood cells, includinggranulocytes) during processing and modulation, culture media of theinvention improves TNC recovery during processing, modulation, orexpansion of the cell population. The culture media additionally enablesmodulation of cells, including activation of the cells by smallmolecules, as may be demonstrated by changes in gene and/or proteinexpression, where such modulation may be impaired or diminished in othercell culture or infusion media.

The culture media may be used in all cell processing steps including,cryopreserving, thawing, resuspension, modulating, expanding, ormaintaining cell populations, particularly populations of cellscomprising hematopoietic stem cells. The culture media may beparticularly useful in processing and manipulating whole blood cellproducts, including whole umbilical cord blood, and mobilized peripheralblood, to prevent cell lysis of, for example, granulocytes and monocytesin the cell population and thereby reduce the occurrence of clumping inthese blood products during processing.

The invention also provides compositions comprising hematopoietic stemcells with high biological activity that provide an improved source ofhematopoietic cells for transplant therapy. The improved cellcompositions have increased therapeutic properties that result inincreased engraftment, increased hematopoietic reconstitution, increasedhoming to the bone marrow, and increased proliferation, in vivo.Accordingly, the improved methods and compositions contemplated hereinmay allow the use of a partial or single cord unit in cord bloodtransplantations.

In various embodiments, methods of preparing a population of cellscomprising hematopoietic cells for transplant therapy are provided. Theimproved methods comprise the use of novel culture media that stabilizeshematopoietic cell populations allowing for modulation and enhancementof the hematopoietic cells, thereby increasing the likelihood ofsuccessful engraftment and hematopoietic reconstitution.

The practice of the invention will employ, unless indicated specificallyto the contrary, conventional methods of chemistry, biochemistry,organic chemistry, molecular biology, microbiology, recombinant DNAtechniques, genetics, immunology, and cell biology that are within theskill of the art, many of which are described below for the purpose ofillustration. Such techniques are explained fully in the literature.See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rdEdition, 2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual(2nd Edition, 1989); Maniatis et al., Molecular Cloning: A LaboratoryManual (1982); Ausubel et al., Current Protocols in Molecular Biology(John Wiley and Sons, updated July 2008); Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, Greene Pub. Associates and Wiley-Interscience; Glover, DNACloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985);Anand, Techniques for the Analysis of Complex Genomes,(Academic Press,New York, 1992); Transcription and Translation (B. Hames & S. Higgins,Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); andHarlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1998).

All publications, patents and patent applications cited herein arehereby incorporated by reference in their entirety.

B. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred embodimentsof compositions, methods and materials are described herein. For thepurposes of the present invention, the following terms are definedbelow.

The articles “a,” “an,” and “the” are used herein to refer to one or tomore than one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “about” or “approximately” refers to aquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length that varies by as much as 30, 25, 20, 25, 10,9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value,number, frequency, percentage, dimension, size, amount, weight orlength. In particular embodiments, the terms “about” or “approximately”when preceding a numerical value indicates the value plus or minus arange of 15%, 10%, 5%, or 1%.

It is understood that in some embodiments, the term “at least” can besubstituted for the term “at least about.”

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. By “consisting of” is meant including, and limitedto, whatever follows the phrase “consisting of.” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory, and that no other elements may be present. By “consistingessentially of” is meant including any elements listed after the phrase,and limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates thatthe listed elements are required or mandatory, but that no otherelements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

Reference throughout this specification to “one embodiment,” “anembodiment,” “a particular embodiment,” “a related embodiment,” “acertain embodiment,” “an additional embodiment,” or “a furtherembodiment” or combinations thereof means that a particular feature,structure or characteristic described in connection with the embodimentis included in at least one embodiment of the present invention. Thus,the appearances of the foregoing phrases in various places throughoutthis specification are not necessarily all referring to the sameembodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

In particular embodiments, the term “resuspension” or “dilution” refersto transferring the thawed cells into a culture medium as contemplatedherein. The thawed cells can be transferred into the same volume ofculture medium or into a larger volume. The thawed cells may be diluted1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or more into a culture medium ascontemplated herein.

The term “ex vivo” refers generally to activities that take placeoutside an organism, such as experimentation or measurements done in oron living tissue in an artificial environment outside the organism,preferably with minimum alteration of the natural conditions. Inparticular embodiments, “ex vivo” procedures involve living cells ortissues taken from an organism and cultured or modulated in a laboratoryapparatus, usually under sterile conditions, and typically for a fewhours or up to about 24 hours, but including up to 48 or 72 hours,depending on the circumstances. In certain embodiments, such tissues orcells can be collected and frozen, and later thawed for ex vivotreatment. Tissue culture experiments or procedures lasting longer thana few days using living cells or tissue are typically considered to be“in vitro,” though in certain embodiments, this term can be usedinterchangeably with ex vivo.

The recitations “ex vivo administration,” “ex vivo treatment,” or “exvivo modulation,” relate generally to medical procedures in which one ormore organs, cells, or tissues are obtained from a living or recentlydeceased subject, optionally purified/enriched, exposed to a treatmentor procedure (e.g., an ex vivo administration step that involvesincubating the cells with a composition or agent of the presentinvention to enhance expansion of particular cells, such ashematopoietic stem or progenitor cells). Cells treated ex vivo may beadministered to the donor or to a different living subject.

Such ex vivo therapeutic applications may also include an optional invivo treatment or procedural step, such as by administering cells withtherapeutic potential one or more times to the living subject. Bothlocal and systemic administration is contemplated for these embodiments,according to well-known techniques in the art and as described elsewhereherein. The amount of therapeutic cells administered to a subject willdepend on the characteristics of that subject, such as general health,age, sex, body weight, and tolerance to drugs, as well as the degree,severity, and type of reaction to the drug and/or cell transplant.

The term “in vivo” refers generally to activities that take place insidean organism, such as cell engraftment, reconstitution, cell homing,self-renewal of cells, and expansion of cells. In one embodiment, theterm “in vivo expansion” refers to the ability of a cell population toincrease in number in vivo. In particular embodiments, the in vivoexpansion include self-renewal and/or proliferation of stem cells.

As used herein, the term “engraftment” refers to the process of a cellintegrating into a location, such as a tissue or site of injury, andbecoming a resident cell in the tissue or at such site. Cells mayengraft in the bone marrow, for instance, or in another location such asa site of injured or ischemic tissue.

In particular embodiments, the term “engraftment” refers to the processof hematopoietic cells locating to the bone marrow and becoming residentcells there. In certain embodiments, engraftment is substantiallyindependent of cell proliferation and independent of reconstitution.“Increased engraftment” occurs when more cells engraft in a samplerelative to the number of cells that engraft in a another sample, suchas a control sample. In some embodiments, increased engraftment occurswhen more cells in a treated sample of cells engrafts compared to thenumber of cells in a non-treated or control sample.

“Engraftment potential” refers to the ability of hematopoietic cells toengraft, and may be assessed by, for example, gene expression thatindicates the cell has the potential for increased engraftment.

As used herein, the term “reconstitution” refers to the ability of oneor more engrafted hematopoietic cells to repopulate or regenerate thehematopoietic system of a subject by giving rise to more progenitors andmore differentiated hematopoietic cell types. In particular embodiments,reconstitution refers to the process of engrafted hematopoietic stemand/or progenitor cells repopulating the hematopoietic system. Long-termreconstitution requires engraftment. “Increased hematopoieticreconstitution” occurs when more of the hematopoietic system isreconstituted with cells in a sample compared to cells in a differentsample, such as a treated sample versus a non-treated sample, which mayonly partially or preferentially reconstitute certain hematopoieticlineages. “Reconstitution potential” refers to the ability ofhematopoietic cells to reconstitute the hematopoietic system, and may beassessed by, for example, gene expression that indicates that the cellhas the potential for increased reconstitution.

“Homing” refers to the ability of HSPCs to localize, i.e., travel, to aparticular area or tissue. Homing may include localization ofadministered HSPCs to the bone marrow or to another location such as asite of injured or ischemic tissue. “Increased homing” occurs when morecells migrate to a target tissue in a sample compared to the number ofcells that migrate to the target tissue in a different sample, such asthe migration seen in a treated sample as compared to an untreatedsample. “Homing potential” the ability of hematopoietic cells to migrateto a target tissue, and may be assessed by, for example, gene expressionthat indicates that the cell has the potential for increased homing.

As used herein, the term “proliferation” refers to an increase in celldivision, either symmetric or asymmetric division of cells. Inparticular embodiments, “proliferation” refers to the symmetric orasymmetric division of stem and/or progenitor. “Increased proliferation”occurs when there is an increase in the number of cells in a treatedsample compared to cells in a non-treated sample. “Proliferationpotential” refers to gene expression characteristics of hematopoieticcells that indicate the cell has the potential for increasedproliferation.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect,including without limitation achieving an improvement or elimination ofsymptoms of a disease. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of achieving an improvement or eliminationof symptoms, or providing a partial or complete cure for a diseaseand/or adverse affect attributable to the disease. “Treatment,” as usedherein, covers any treatment of a disease in a mammal, particularly in ahuman, and includes: (a) preventing the disease from occurring in asubject which may be predisposed to the disease but has not yet beendiagnosed as having it; (b) inhibiting the disease, i.e., arresting itsdevelopment; (c) relieving the disease, e.g., causing regression of thedisease, e.g., to completely or partially eliminate symptoms of thedisease; and (d) restoring the individual to a pre-disease state, e.g.,reconstituting the hematopoietic system.

By “enhance” or “promote,” or “increase” or “activate” refers generallyto the ability of an agent to produce or cause a greater physiologicalresponse (i.e., downstream effects) in a cell, as compared to theresponse caused by either vehicle or a control molecule/composition,e.g., increased engraftment or reconstitution of hematopoietic stem andprogenitor cells and increased in vivo stem cell expansion. A measurablephysiological response may include an increase in hematopoietic stem andprogenitor cell engraftment, reconstitution, viability, homing,self-renewal, and/or expansion, among others apparent from theunderstanding in the art and the description herein. In one embodiment,the measurable physiological response includes increased expression of aplurality of genes that are markers for therapeutic potential ofhematopoietic cells, compared to the expression of the genes in areference sample (e.g., control or untreated cells). An “increased” or“enhanced” amount is typically a “statistically significant” amount, andmay include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including allintegers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7.1.8, etc.) the response produced by vehicle (the absence of an agent) ora control composition.

By “decrease” or “lower,” or “lessen,” or “reduce,” or “abate” refersgenerally to the ability of an agent to produce or cause a lesserphysiological response (i.e., downstream effects) in a cell, as comparedto the response caused by either vehicle or a controlmolecule/composition, e.g., decreased gene expression. In oneembodiment, the decrease can be a decrease in gene expression or adecrease in cell signaling that normally is associated with a reductionof cell viability. An “decrease” or “reduced” amount is typically a“statistically significant” amount, and may include an decrease that is1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times(e.g., 500, 1000 times) (including all integers and decimal points inbetween and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the responseproduced by vehicle (the absence of an agent) or a control composition.

By “maintain,” or “preserve,” or “maintenance,” or “no change,” or “nosubstantial change,” “no substantial increase,” or “no substantialdecrease” refers generally to the ability of a agent to produce or causea comparable physiological response (i.e., downstream effects) in acell, as compared to the response caused by either vehicle or a controlmolecule/composition (reference response). A comparable response is onethat is not significantly different or measurably different from thereference response.

The “therapeutic potential” of a cell refers to the therapeutic qualityof the cell, the cell's ability to provide a therapeutic benefit whenadministered to a subject. In particular embodiments, the therapeuticpotential of a cell can be measured, quantified, determined, identified,or validated by increased expression of a plurality of genes and/or bythe presence of a particular gene expression signature that indicatesthe cell's therapeutic potential. In one embodiment, therapeuticpotential refers to a cell's ability to home and engraft to a particulartissue, organ, or site of injury. In a particular embodiment,therapeutic potential refers to a cell's ability to reconstitute thehematopoietic system of a subject. In a certain embodiment, therapeuticpotential refers to a cell's ability undergo self-renewal in vivo onceadministered to a subject. In particular embodiments, the terms“therapeutic cell,” “cell with therapeutic potential,” and “cell havingtherapeutic potential” are used interchangeably.

In particular embodiments, cells that have increased expression of aplurality of genes and/or a particular gene expression signature have“sufficient therapeutic potential.” The therapeutic potential of thecells is sufficient is they have the ability to engraft, the ability toreconstitute cell lineages, and/or the ability to proliferate whenadministered to a subject.

In certain embodiments, cells with therapeutic potential comprise uniqueor substantially unique gene and/or protein expression. The cellscomprising unique or substantially unique expression are deemed to havetherapeutic potential. In particular embodiments, the phrase “expressionof a plurality of genes” refers to gene expression, the expression ofmRNA. In other embodiments, the phrase “expression of a plurality ofgenes” refers to the level of protein expression.

A “plurality” of genes refers to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56,5 7, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300,400, 500 or more genes, including any intervening number of genes.

As used herein, the term “gene expression profile,” “gene expressionsignature,” “gene expression panel,” “gene panel,” or “gene signature”refers to the levels of expression of a plurality (i.e., more than one)of genes measured for the same sample, i.e., a population of cells. Agene expression signature may be defined so as to identify a group ofgenes “signature genes” or a “plurality of genes” that serves todistinguish the therapeutic cells or cells having therapeutic potentialfrom existing cells in the art and/or control, vehicle, or non-treatedcells.

A “signature gene”, as used herein, means any gene in a group ofsignature genes or plurality of genes. For clarity, signature genes donot include housekeeping genes.

“Gene expression” as used herein refers to the relative levels ofexpression and/or pattern of expression of a gene in a biologicalsample, such as the stem and progenitor cells, or population of cellscomprising stem or progenitor cells. In particular embodiments, the stemor progenitor cells are hematopoietic stem and progenitor cells.

“Genetic modification” refers to a temporary or permanent modificationof a cell's genome, for example by insertion of a polynucleotidesequence in a viral or plasmid vector, or by homologous recombination ornon-homologous end joining.

As used herein, the term “gene therapy” refers to the introduction of apolynucleotide into a cell that restores, corrects, or modifies the geneand/or expression of the gene. In particular embodiments, the genetherapy modifies the genome, and in other embodiments, the gene therapyis episomal.

As used herein, the phrases “detecting expression,” “determiningexpression,” and “measuring expression” refer to determining thequantity or presence of an RNA transcript or its expression product of agene. Methods for detecting expression of genes, that is, geneexpression profiling, include methods based on hybridization analysis ofpolynucleotides, methods based on sequencing of polynucleotides,immunohistochemistry methods, and proteomics-based methods. The methodsgenerally detect expression products (e.g., mRNA) of the genes ofinterest. In some embodiments, PCR-based methods, such as reversetranscription PCR (RT-PCR) (Weis et al., TIG 8:263-64, 1992), andarray-based methods such as microarray (Schena et al., Science270:467-70, 1995) are used.

General methods for RNA extraction are well known in the art and aredisclosed in standard textbooks of molecular biology, including Ausubelet al., ed., Current Protocols in Molecular Biology, John Wiley & Sons,New York 1987-1999. In particular, RNA isolation can be performed usinga purification kit, a buffer set and protease from commercialmanufacturers, such as Qiagen (Valencia, Calif.), according to themanufacturer's instructions. For example, total RNA from cells inculture can be isolated using Qiagen RNeasy mini-columns. Isolated RNAcan be used in hybridization or amplification assays that include, butare not limited to, PCR analyses and probe arrays. One method for thedetection of RNA levels involves contacting the isolated RNA with anucleic acid molecule (probe) that can hybridize to the mRNA encoded bythe gene being detected. The nucleic acid probe can be, for example, afull-length cDNA, or a portion thereof, such as an oligonucleotide of atleast 7, 15, 30, 60, 100, 250, or 500 nucleotides in length andsufficient to specifically hybridize under stringent conditions to anintrinsic gene of the present invention, or any derivative DNA or RNA.Hybridization of an mRNA with the probe indicates that the intrinsicgene in question is being expressed.

An alternative method for determining the level of gene expression in asample involves the process of nucleic acid amplification, for example,by RT-PCR (U.S. Pat. No. 4,683,202), ligase chain reaction (Barany,Proc. Natl. Acad. Sci. USA 88:189-93, 1991), self sustained sequencereplication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-78,1990), transcriptional amplification system (Kwoh et al., Proc. Natl.Acad. Sci. USA 86:1173-77, 1989), Q-Beta Replicase (Lizardi et al.,Bio/Technology 6:1197, 1988), rolling circle replication (U.S. Pat. No.5,854,033), or any other nucleic acid amplification method, followed bythe detection of the amplified molecules using techniques well known tothose of skill in the art.

Numerous different PCR or qPCR protocols are known in the art andexemplified herein below and can be directly applied or adapted for useusing the cell potency assays contemplated herein to determinetherapeutic potential. Quantitative PCR (qPCR) (also referred asreal-time PCR) is preferred under some circumstances because it providesnot only a quantitative measurement, but also reduced time andcontamination. In some instances, the availability of full geneexpression profiling techniques is limited due to requirements for freshfrozen tissue and specialized laboratory equipment, making the routineuse of such technologies difficult in a clinical setting. As usedherein, “quantitative PCR (or “real time qPCR”) refers to the directmonitoring of the progress of PCR amplification as it is occurringwithout the need for repeated sampling of the reaction products. Inquantitative PCR, the reaction products may be monitored via a signalingmechanism (e.g., fluorescence) as they are generated and are trackedafter the signal rises above a background level but before the reactionreaches a plateau. The number of cycles required to achieve a detectableor “threshold” level of fluorescence varies directly with theconcentration of amplifiable targets at the beginning of the PCRprocess, enabling a measure of signal intensity to provide a measure ofthe amount of target nucleic acid in a sample in real time.

C. Culture Media

In various embodiments, improved culture media for the manipulation ofblood cell products, are contemplated. The culture media increases totalnuclear cell (TNC) count and cell viability of blood cell productsduring processing, including during cryopreservation, thawing,resuspension, culturing, or manipulation, including in blood cellproducts that have been thawed and/or modulated or expanded in vitro orex vivo by one or more agents. The cell culture media also increasesstability of the blood cell product by stabilizing the membranes ofapoptosing cells, thereby decreasing cell lysis, preventing cell debrisaggregation, and preventing the release of intracellular components thatcan inhibit cellular processes. In one embodiment, the cell culturemedia comprises a stock cell culture media used for culturing stem cellsthat is supplemented with a polysaccharide. In some embodiments, thecell culture media comprises a chemically defined stock basal media,such as any defined basal media suitable for supporting the maintenance,growth, and/or differentiation of stem cells, such as conventional humanembryonic stem cell media, that is supplemented with a polysaccharide.In particular embodiments, a culture medium comprises polysaccharides,human serum albumin (HSA), and a chemically defined medium.

In some embodiments, the culture media are suitable for one or more offreezing, thawing, resuspension, processing or purification, modulationwith one or more agents, or expansion of hematopoietic cells, e.g.,HSPCs. In certain embodiments, the culture media are suitable forthawing, resuspension, processing or purification, modulation,expansion, and administration of hematopoietic cells to a subject. Infurther embodiments, the culture media are suitable for thawing,resuspension, processing or purification, modulation, and expansion ofhematopoietic cells and the hematopoietic cells are subsequently washedwith a pharmaceutically acceptable cell culture medium foradministration to a subject.

1. Polysaccharides

In one embodiment, a cell culture medium or composition comprises one ormore low molecular weight polysaccharides. A “polysaccharide” refers toany of a large class of long-chain sugars composed of monosaccharides.Because the chains may be unbranched or branched and the monosaccharidesmay be of one, two, or occasionally more kinds, polysaccharides can becategorized in various ways. In particular embodiments, cell culturemedia and compositions comprise about 1% to about 20% polysaccharide,about 1% to about 15% polysaccharide, about 1% to about 10%polysaccharide, or about 5% to about 10% polysaccharide. In certainembodiments, the culture media and compositions may comprise at leastabout 2%, at least about 3%, at least about 4%, at least about 5%, atleast about 6%, at least about 7%, at least about 8%, at least about 9%,at least about 10%, at least about 11%, at least about 12%, at leastabout 13%, at least about 14% at least about 15%, at least about 16%, atleast about 17%, at least about 18%, at least about 19%, or at leastabout 20% polysaccharide.

Without wishing to be bound to any particular theory, it is contemplatedthat increasing the polysaccharide content in cell culture mediumstabilizes the membranes of the cells after being thawed and preventscell lysis in apoptosing cells, thereby increasing the TNC count andhematopoietic stem and progenitor cell (HSPC) viability of the bloodcell product. In certain preferred embodiments, a culture medium orcomposition comprises a polysaccharide selected from the groupconsisting of a dextran and a starch.

Dextrans

In various embodiments, a cell culture medium or composition comprisesone or more low molecular weight dextrans. Dextrans are polysaccharidescomposed of an α-D-1,6-glucose-linked glucan with side-chains 1-3 linkedto the backbone units of the dextran biopolymer. The degree of branchingis approximately 5%. The branches are mostly 1-2 glucose units long.Dextran can be obtained from fermentation of sucrose-containing media byLeuconostoc mesenteroides B512F. Dextrans are isotonic and can be storedat room temperature.

Illustrative examples of low molecular weight dextrans include dextranswith a molecular weight of about 1000 Da (dextran-1), about 10,000 Da(dextran-10), about 20,000 Da (dextran-20), about 30,000 Da(dextran-30), about 40,000 Da (dextran-40), or about 50,000 Da(dextran-50).

In particular embodiments, a cell culture medium or compositioncomprises at least 1.0%, at least 2.0%, at least 3.0%, at least 4.0%, atleast 5.0%, at least 6.0%, at least 7.0%, at least 8.0%, at least 9.0%,at least 10.0%, at least 11.0%, at least 12.0%, at least 13.0%, at least14.0%, at least 15.0%, at least 16.0%, at least 17.0%, at least 18.0%,at least 19.0%, or at least 20.0% dextran. In certain embodiments, acell culture medium or composition comprises about 1% to about 20%dextran, about 2.5% to about 15% dextran, about 5% to about 12.5%dextran, or about 5% to about 10% dextran. In one embodiment, thedextran is one or more of dextran-1, dextran-10, dextran-20, dextran-30,dextran-40, or dextran-50. In a particular embodiment, the dextran isdextran-40.

Hydroxyethyl Starch (HES)

In various embodiments, a cell culture medium or composition comprisesone or more low molecular weight starches. A “starch” refers topolysaccharide that is a white odorless tasteless granular or powderycomplex carbohydrate (C₆H₁₀O₅)_(x) that is the chief storage form ofcarbohydrate in plants. In particular embodiments, a cell culture mediumcomprises a hydroxyethyl starch (HES).

HES is the parent name of a polymeric molecule made from a waxy speciesof either maize or sorghum and is composed primarily of amylopectin(98%). It is a highly branched polysaccharide closely resemblingglycogen, formed by the reaction between ethylene oxide and amylopectinin the presence of an alkaline catalyst. The molecular weight and molarsubstitution can be adjusted by the degree of substitution of hydroxylgroups with hydroxyethyl groups at the C₂, C₃ and C₆ positions on theglucose molecule. The greater the substitution on position C₂ inrelation to C₆ (C₂:C₆ ratio), the greater the half-life.

The number-averaged molecular weight (Mn) is the arithmetic mean of themolecular weights of the polymers in solution. Weight-averaged molecularweight (Mw) is the sum of the number of molecules at each number dividedby the total of all molecules. This weight is generally larger whenlarger polymers are present in solution. The classification of differentHES products includes the ratio of the Mw and the degree ofsubstitution.

Illustrative examples of HES products include, but are not limited tohetastarch (0.7 degree substitution), hexastarch (0.6 degreesubstitution), pentastarch (0.5 degree substitution), and tetrastarch(0.4 degree substitution).

In particular embodiments, a cell culture medium or compositioncomprises about 1.0%, at least 2.0%, at least 3.0%, at least 4.0%, atleast 5.0%, at least 6.0%, at least 7.0%, at least 8.0%, at least 9.0%,at least 10.0%, at least 11.0%, at least 12.0%, at least 13.0%, at least14.0%, at least 15.0%, at least 16.0%, at least 17.0%, at least 18.0%,at least 19.0%, or at least 20.0% HES. In certain embodiments, the HESis selected from the group consisting of: hetastarch, hexastarch,pentastarch, and tetrastarch. In particular media formulations, a cellculture medium comprises one or more starches selected from the groupconsisting of: hetastarch, hexastarch, pentastarch, and tetrastarch. Inone embodiment, a cell culture medium or composition comprises about2.5% to about 12.5% HES, about 2.5% to about 10% HES, about 5% to about12.5% HES, or about 5% to about 10% HES.

In one particular embodiment, a cell culture medium comprises both adextran and a HES at a total starch concentration of 1.0%, at least2.0%, at least 3.0%, at least 4.0%, at least 5.0%, at least 6.0%, atleast 7.0%, at least 8.0%, at least 9.0%, at least 10.0%, at least11.0%, at least 12.0%, at least 13.0%, at least 14.0%, at least 15.0%,at least 16.0%, at least 17.0%, at least 18.0%, at least 19.0%, or atleast 20.0%.

2. Human Serum Albumin (HSA)

Human serum albumin (HSA) is the most abundant protein in human plasmawith a molecular weight of 66,437 Da (based on amino acid composition).Commercial preparations contain varying degrees of post-translationalmodifications and genetic variants with molecular weight componentsmainly in the range of 66,437 to 66,600 Da.

In various embodiments, a cell culture medium or composition comprisesHSA. In particular embodiments, a cell culture medium or compositioncomprises about 1.0%, about 1.25%, about 1.5%, about 1.75%, about 2.0%,about 2.25%, about 2.5%, about 2.75%, about 3.0%, about 3.25%, about3.5%, about 3.75%, about 4.0%, about 4.25%, about 4.5%, about 4.75%,about 5.0%, about 5.25%, about 5.5%, about 5.75%, about 6.0%, about6.25%, about 6.5%, about 6.75%, about 7.0%, about 7.25%, or about 7.5%HSA.

In another embodiment, a cell culture medium or composition comprisesabout 2.0% to about 7.5%, about 2.5% to about 6%, or about 4.0% to about6%, or about 4% to about 5% HSA.

3. Chemically Defined Cell Culture Media

A “chemically defined medium” refers to a growth medium suitable for thein vitro or ex vivo cell culture of human or animal cells in which allof the chemical components are known. In a particular embodiment, achemically defined medium is entirely free of animal-derived componentsand does not contain either fetal bovine serum, bovine serum albumin orhuman serum albumin as these products are derived from bovine or humansources and contain complex mixes of albumins and lipids. However, incertain embodiments, a composition may comprise a chemically definedmedia and one or more of the foregoing types of sera or additionalagents, e.g., cytokines, growth factors, prostaglandin pathway agonists,and glucocorticoids.

A defined and humanized medium for the culture and proliferation and/ormaintenance of human hematopoietic cells typically includes salts,vitamins, a source of glucose, minerals and amino acids. In particularembodiments, the defined medium may be supplemented with human serum orwith a serum replacement. The serum replacement can be a commerciallyavailable product sold for that purpose or can be a formulated mixtureof protein, such as serum albumin, vitamins, salts, minerals, atransferrin or transferrin substitute, and insulin or an insulinsubstitute. This serum replacement component may also be supplementedwith selenium. In one embodiment, a culture medium comprises a definedmedium that is supplemented with human serum albumin, vitamins,antioxidants, trace minerals, specific lipids, and cloned growthfactors.

In particular embodiments, the defined medium is also a pharmaceuticallyacceptable cell-culture medium. Such compositions are suitable foradministration to human subjects. Generally speaking, any medium thatsupports the maintenance, growth, and/or health of the hematopoieticcells of the invention are suitable for use as a pharmaceutical cellculture medium. In particular embodiments, the pharmaceuticallyacceptable cell culture medium is a serum free medium or chemicallydefined medium.

One illustrative example of a pharmaceutically acceptable cell culturemedium includes Calcium Chloride Anhydrous CaCl3 (158.695 mg/L); CupricSulfate CuSO4 5H2O (0.000654 mg/L); Ferric Nitrate Fe(NO3) 9H2O (0.0751mg/L); Ferric Sulfate FeSO47H2O (0.0209 mg/L); Potassium Chloride KCl(306.969 mg/L); Magnesium Chloride MgCl2 (14.418 mg/L); MagnesiumSulfate MgSO4 (63.237 mg/L); Sodium Chloride NaCl (5021.73 mg/L); SodiumBicarbonate NaHCO4 (1100 mg/L); Sodium Phosphate Monobasic NaH2PO4H2O(93.964 mg/L); Sodium Phosphate dibasic Na2HPO4 7H2O (35.753 mg/L); ZincSulfate ZnSO4 7H2O (0.217 mg/L); D-Glucose (Dextrose) (3836.3 mg/L);Phenol Red (8.127 mg/L); HEPES (3099.505 mg/L); Na Hypoxanthine (1.203mg/L); Linoleic acid (0.0211 mg/L); DL-68-Thioctic Acid (0.0528 mg/L);Sodium Putrescine 2HCl (0.0407 mg/L); Putrescine 8 Sodium Selenite(2.5×10-6 mg/L); Sodium Pyruvate (40.1885 mg/L); Alanine (3.24 mg/L);Arginine HCl (116.255 mg/L); Asparagine (4.19 mg/L); Aspartic acid(3.347 mg/L); Cysteine H2O (9.445 mg/L); Cysteine 2HCl (15.752 mg/L);Glutamic acid (3.7 Glutamine (293.55 mg/L); Glycine (24.439 mg/L);Histidine HCl H2O (36.847 mg/L); Isoleucine (79.921 mg/L); Leucine(82.227 mg/L); Lysine HCl (118.937 mg/L); Methionine (23.679 mg/L);Phenylalanine (50.861 mg/L); Proline (12.564 mg/L); Serine (34.214mg/L); Threonine (74.408 mg/L); Tryptophan (12.54 mg/L); Tyrosine 2Na+2H2O (64.086 mg/L); Valine (73.606 mg/L); Biotin (0.00176 mg/L);D-Calcium panthenate (3.127 mg/L); Choline chloride (6.52 mg/L); Folicacid (3.334 mg/L); i-Inositol (9.904 mg/L); Niacinamide (3.079mg/L);Pyridoxine HCl (3.022 mg/L); Riboflavine (0.31 mg/L); Thiamine HCl(3.092 mg/L); Thymidine (0.183 mg/L); and Vitamin B12 (0.512 mg/L).

Illustrative examples of chemically defined cell culture media suitablefor use in the particular embodiments include, but are not limited toSTEMSPAN-ACF, STEMSPAN-H3000, STEMSPAN-SFEM, STEMLINE II, STEMPRO 34,STEMSVIVO, Iscove's modified Dulbecco's medium (IMDM), Dulbecco'smodified Eagle medium (DMEM), Roswell Park Memorial Institute medium(RPMI) 1640 medium, McCoy's 5A medium, minimum essential medium alphamedium (alpha-MEM), basal medium Eagle (BME), Fischer's medium,medium199, F-12K nutrient mixture medium (Kaighn's modification, F-12K),and X-vivo 20.

Illustrative examples of cytokines and/or hematopoietic cell growthfactors include, but are not limited to flt3-ligand (FLT3),thrombopoietin (TPO), stem cell factor (SCF), epidermal growth factor(EGF), transforming growth factor-beta (TGF-(3), basic fibroblast growthfactor (bFGF), interleukin-3 (IL3), interleukin-6 (IL6), andinterleukin-9 (IL9).

D. Compositions

In various embodiments, compositions comprising a blood cell product orpartially isolated or purified hematopoietic cells and an improvedculture media for the manipulation of blood cell products arecontemplated. The cells of the composition have increased cell viabilityand can tolerate freeze/thaw processing, and in vitro or ex vivomodulation or expansion by one or more agents. The compositions alsocomprise apoptosing cells with increased stability, thereby decreasingcell lysis, preventing cell debris aggregation, and preventing therelease of intracellular components that can inhibit subsequent cellularprocesses.

In particular embodiments, the compositions comprise a blood cellproduct or a population of isolated or purified hematopoietic cells. Insome embodiments, the compositions are suitable for one or more offreezing, thawing, resuspension, processing or purification, modulationwith one or more agents, or expansion. In certain embodiments, thecompositions are suitable for thawing, resuspension, processing orpurification, modulation, expansion, and administration to a subject. Infurther embodiments, the compositions are suitable for thawing,resuspension, processing or purification, modulation, and expansion, andare washed with a pharmaceutically acceptable cell culture medium foradministration to a subject.

In particular embodiment, compositions comprise blood cell products orpopulations of hematopoietic cells that are HLA typed and may be matchedor partially matched to a specific patient for transplantation.

At a minimum, HLA typing of the hematopoietic cell population isperformed for six HLA loci, HLA-A, -B, and -DR, for example, at lowresolution/split antigen level.

In various embodiments, the blood cell product or population ofhematopoietic cells comprises haplotyped hematopoietic stem orprogenitor cells. In some embodiments, the population of cellscomprising the therapeutic composition is HLA typed based on HLA-A,HLA-B, HLA-C, and HLA-DRB1. In particular embodiments, the population ofcells is HLA typed based on the group consisting of HLA-DRB3/4/5,HLA-DQB1, and DPB1. In some embodiments, the population of cellscomprising the therapeutic composition is matched with a specific humanpatient. In some embodiments, the population of HLA haplotyped cells has4 out of 6 HLA matches with a specific human subject. HLA matching maybe based on alleles or antigens, and combinations thereof. In someembodiments, the population of HLA haplotyped cells is a partialmismatch with a specific human subject, such as the subject to which thetherapeutic composition is administered.

1. Blood Cell Products

In particular embodiments, compositions may comprise blood cell productsor a portion thereof. Suitable blood cell products may be obtained froma blood bank or directly from a donor or patient. In particularembodiments, a composition comprises one or more units of a blood cellproduct. Suitable sources of blood cell products include, but are notlimited to the bone marrow, the umbilical cord, umbilical cord blood,placental blood, the placenta, fetal blood, fetal liver, fetal spleen,Wharton's jelly, and mobilized peripheral blood.

In one embodiment, the blood cell product is umbilical cord blood. Asused herein, the term “cord blood,” “whole cord blood,” “whole umbilicalcord blood,” or “umbilical cord blood” relates generally to therelatively small amount of blood (up to about 180 mL) from a newbornbaby that returns to the neonatal circulation if the umbilical cord isnot prematurely clamped. Cord blood is rich in HSPCs, and may beharvested and stored for later use according to techniques known in theart (see, e.g., U.S. Patent Nos. 7,147,626 and 7,131,958, hereinincorporated by reference for such methodologies). In one embodiment,whole cord blood does not include red blood cells and/or plasma.

In particular embodiments, a blood cell product requires a sufficientamount of hematopoietic cells for use in therapeutic applications.Increasing the amount of hematopoietic cells in a blood cell product maycomprise a step of mobilizing hematopoietic stem and progenitor cells ina subject. A sufficient amount of hematopoietic stem and progenitorcells in the blood cell products contemplated for use in particularembodiments comprise at least about 1×10³ HSPCs, at least about 1×10⁴HSPCs, at least about 1×10⁵ HSPCs, at least about 1×10⁶ HSPCs, at leastabout 1×10⁷ HSPCs, at least about 1×10⁸ HSPCs, at least about 1×10⁹HSPCs, at least about 1×10¹⁰ HSPCs, at least about 1×10¹¹ HSPCs, atleast about 1×10¹² HSPCs, at least about 1×10¹³ HSPCs, at least about1×10¹⁴ HSPCs, or at least about 1×10¹⁵ HSPCs.

“Hematopoietic stem cell mobilization” refers to the release of stemcells from the bone marrow or another tissue comprising hematopoieticstem and progenitor cells into the peripheral blood circulation for thepurpose of leukapheresis, prior to stem cell transplantation. Byincreasing the number of stem cells harvested from the donor, the numberof stem cells available for therapeutic applications can besignificantly improved. Hematopoietic growth factors, e.g., granulocytecolony stimulating factor (G-CSF) or chemotherapeutic agents often areused to stimulate the mobilization. Commercial stem cell mobilizationdrugs exist and can be used in combination with G-CSF to mobilizesufficient quantities of hematopoietic stem and progenitor cells fortransplantation into a subject. For example, G-CSF and Mozobil™ (GenzymeCorporation) can be administered to a donor in order to harvest asufficient number of hematopoietic cells for transplantation. Othermethods of mobilizing hematopoietic stem and progenitor cells would beapparent to one having skill in the art.

2. Hematopoietic Cells

In various embodiments, compositions contemplated herein comprise apurified or isolated population of cells comprising hematopoietic cells.As used herein, the term “isolated” refers to material that is removedfrom its original environment. For example, an “isolated population ofcells,” an “isolated source of cells,” or “isolated HSPCs” and the like,as used herein, refer to in vitro or ex vivo separation of one or morecells from their natural cellular environment, and from association withother components of the tissue or organ, i.e., it is not significantlyassociated with in vivo substances. In particular embodiments, acomposition comprises a population of hematopoietic stem or progenitorcells.

In particular embodiments, compositions comprise cells that areautologous/autogeneic (“self') or non-autologous (“non-self,” e.g.,allogeneic, syngeneic or xenogeneic) cells. “Autologous,” as usedherein, refers to cells from the same subject. “Allogeneic,” as usedherein, refers to cells of the same species that differ genetically tothe cell in comparison. “Syngeneic,” as used herein, refers to cells ofa different subject that are genetically identical to the cell incomparison. “Xenogeneic,” as used herein, refers to cells of a differentspecies to the cell in comparison. In preferred embodiments, the cellsof the invention are allogeneic.

In various embodiments, the use of stem cells is preferred because theyhave the ability to differentiate into the appropriate cell types whenadministered to a particular biological niche, in vivo. The term “stemcell” refers to a cell which is an undifferentiated cell capable of (1)long term self -renewal, or the ability to generate at least oneidentical copy of the original cell, (2) differentiation at the singlecell level into multiple, and in some instance only one, specializedcell type and (3) of in vivo functional regeneration of tissues. As usedherein, the term “progenitor” or “progenitor cells” refers to cells thathave the capacity to self-renew and to differentiate into more maturecells. Progenitor cells have a reduced potency compared to pluripotentand multipotent stem cells.

As used herein, the term “hematopoietic stem and progenitor cell” or“HSPC” refers to a cell identified by the presence of the antigenicmarker CD34 (CD34⁺) and are therefore characterized as CD34⁺ cells, andpopulations of such cells. In particular embodiments, the term “HSPC”refers to a cell identified by the presence of the antigenic marker CD34(CD34⁺) and the absence of lineage (Lin) markers and are thereforecharacterized as CD34⁺/Lin(−) cells, and populations of such cells. Itis recognized that the population of cells comprising CD34⁺ and/orLin(−) cells also includes hematopoietic progenitor cells. In oneembodiment, a composition comprises a population of isolated CD34⁺cells.

Hematopoietic stem cells are multipotent stem cells that give rise toall the blood cell types of an organism, including myeloid (e.g.,monocytes and macrophages, neutrophils, basophils, eosinophils,erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoidlineages (e.g., T-cells, B-cells, NK-cells), and others known in the art(See Fei, R., et al., U.S. Patent No. 5,635,387; McGlave, et al., U.S.Patent No. 5,460,964; Simmons, P., et al, U.S. Patent No. 5,677,136;Tsukamoto, et al., U.S. Patent No. 5,750,397; Schwartz, et al., U.S.Patent No. 5,759,793; DiGuisto, et al., U.S. Patent No. 5,681,599;Tsukamoto, et al., U.S. Patent No. 5,716,827). Hematopoietic progenitorcells (HSCs) give rise to committed hematopoietic progenitor cells(HPCs) that are capable of generating the entire repertoire of matureblood cells over the lifetime of an organism.

As used herein, the term “granulocytes” refers to a category of whiteblood cells characterized by the presence of granules in theircytoplasm. Granulocytes are often called polymorphonuclear leukocytes(PMN or PML) because of the varying shapes of the nucleus, which isusually lobed into three segments. Although the most abundant type ofgranulocytes are neutrophil granulocytes, the term “granulocyte”includes neutrophil granulocytes, eosinophil granulocytes, and basophilgranulocytes.

In particular embodiments, HPSCs can be provided as a highly purifiedHSPC population (a homogenous population), or as a composition thatcomprises from 0.01% to about 100% of HSPCs (a heterogeneouspopulation). Populations of cells comprising hematopoietic stem andprogenitor cells include bone marrow cells, umbilical cord blood cells,placental blood cells, mobilized peripheral blood cells, hematopoieticstem cells, or hematopoietic progenitor cells. In particularembodiments, the number of HSPCs in a population of hematopoietic cellscan be increased by mobilizing the stem and progenitor cells in thedonor, as discussed supra.

In one embodiment, a composition comprises the amount of HSPCs in apartial or single cord of blood, or is at least 0.1×10⁵ cells/kg ofbodyweight, at least 0.5×10⁵ cells/kg of bodyweight, at least 1×10⁵cells/kg of bodyweight, at least 5×10⁵ cells/kg of bodyweight, at least10×10⁵ cells/kg of bodyweight, at least 0.5×10⁶ cells/kg of bodyweight,at least 0.75×10⁶ cells/kg of bodyweight, at least 1×10⁶ cells/kg ofbodyweight, at least 1.25×10⁶ cells/kg of bodyweight, at least 1.5×10⁶cells/kg of bodyweight, at least 1.75×10⁶ cells/kg of bodyweight, atleast 2×10⁶ cells/kg of bodyweight, at least 2.5×10⁶ cells/kg ofbodyweight, at least 3×10⁶ cells/kg of bodyweight, at least 4×10⁶cells/kg of bodyweight, at least 5×10⁶ cells/kg of bodyweight, at least10×10⁶ cells/kg of bodyweight, at least 15×10⁶ cells/kg of bodyweight,at least 20×10⁶ cells/kg of bodyweight, at least 25×10⁶ cells/kg ofbodyweight, or at least 30×10⁶ cells/kg of bodyweight.

In particular embodiments, a composition comprises about 1×10³ HSPCs, atleast about 1×10⁴ HSPCs, at least about 1×10⁵ HSPCs, at least about1×10⁶ HSPCs, at least about 1×10⁷ HSPCs, at least about 1×10⁸ HSPCs, atleast about 1×10⁹ HSPCs, at least about 1×10¹⁰ HSPCs, at least about1×10¹¹ HSPCs, at least about 1×10¹² HSPCs, at least about 1×10¹³ HSPCs,at least about 1×10¹⁴ HSPCs, or at least about 1×10¹⁵ HSPCs. In oneembodiment, the HSPCs are CD34⁺.

In particular embodiments, a composition comprises a population of cellsthat is about 95% to about 100% HSPCs. In some embodiments, thepopulation of cells comprises less than about 0.1%, 0.5%, 1%, 2%, 5%,10%, 15%, 20%, 25%, or 30% HSPCs. The population of cells in someembodiments comprises less than about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%,20%, 25%, or 30% HSPCs. In other embodiments, the population of cells isabout 0.1% to about 1%, about 1% to about 3%, about 3% to about 5%,about 10%-about 15%, about 15%-20%, about 20%-25%, about 25%-30%, about30%-35%, about 35%-40%, about 40%-45%, about 45%-50%, about 60%-70%,about 70%-80%, about 80%-90%, about 90%-95%, or about 95% to about 100%HSPCs.

In particular embodiments, the population of cells is about 0.1% toabout 1%, about 1% to about 3%, about 3% to about 5%, about 10%-about15%, about 15%-20%, about 20%-25%, about 25%-30%, about 30%-35%, about35%-40%, about 40%-45%, about 45%-50%, about 60%-70%, about 70%-80%,about 80%-90%, about 90%-95%, or about 95% to about 100% HSPCs.

In various embodiments, the cells are not genetically modified cells. Inother embodiments, the cells are genetically modified, such as byintroducting of a polynucleotide, such as, for example a retroviral orlentiviral vector comprising a protein coding gene sequence. In someembodiments, the cell is genetically modified to correct a geneticdefect and in other embodiments, the cell is genetically modified toincrease or decrease production of a wild-type or mutant protein.Polynucleotides used to increase expression of a protein in a cell maycomprise polynucleotide sequences to direct appropriate expression inthe cell and a polynucleotide encoding the polypeptide sequence.Polynucleotides used to decrease expression of a protein in a cell maycomprise polynucleotide sequences that target polynucleotides encodingthe wild type polypeptide sequence for degradation.

3. Enhanced Hematopoietic Cells

In particular embodiments, compositions comprise human hematopoieticstem and progenitor cells wherein the stem cells have been contacted exvivo with one or more agents capable of increasing the therapeuticproperties of the cell. In one embodiment, human hematopoietic stem andprogenitor cells have been contacted ex vivo with one or more agentsthat increase CXCR4 gene expression in the cells. In one preferredembodiment, the gene expression of CXCR4 is increased in the treatedhuman hematopoietic stem cells at least about 2, 3, 4, 5, 10, 15, 20, or30 fold compared to non-contacted hematopoietic stem and progenitorcells or cells treated with a vehicle control. “Enhanced hematopoieticstem and progenitor cell” or “enhanced HSPC” refers to a HSPC treated exvivo with one or more agents that increase CXCR4 gene expression in thecell at least about 2, 3, 4, 5, 10, 15, 20, or 30 compared to control,vehicle or untreated cells.

As used herein, a “non-contacted,” “untreated,” or “control” cell is acell that has not been treated, e.g., cultured, contacted, or incubatedwith an agent other than a control agent. Blood cell products orhematopoietic cells contacted with DMSO (a control agent), or contactedwith another vehicle (10% dextran, 5%HSA, and 0.9% saline) are controlcells.

The HSPCs of the invention are identified and are characterized by, agene expression profile indicating high levels of CXCR4 expression. TheHSPCs can also be characterized based upon increased CXCR4 geneexpression and increased cell surface expression of CXCR4 polypeptide.In certain embodiments, the CXCR4 gene expression in the HSPCs of theinvention is increased by at least 2, 3, 4, 5, 10, 15, 20, or 30 foldcompared to the expression of CXCR4 in non-contacted cells. In certainembodiments, the CXCR4 gene expression in the HSPCs of the invention isincreased by at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, or 100 fold compared to the expression of CXCR4 in non-contactedcells.

In particular embodiments, CXCR4 gene expression in the HSPCs isincreased by about 30 to about 80 fold compared to untreated HSPCs. Infurther embodiments, CXCR4 gene expression in the HSPCs is increased byabout 40 to about 80 fold, about 50 to about 80 fold, about 60 to about80 fold, or about 50 to about 70 fold, compared to untreated HSPCs.

CXCR4 gene expression or the gene expression signature of the treatedHSPCs or an aliquot thereof may be determined after the cells aretreated with one or more agents. For example, HSPCs may be treated exvivo with one or more agents, washed to remove the agent(s), and thegene expression analyzed from a portion of the cells without furtherincubation of the cells.

An illustrative group of genes, e.g., “signature genes” for use inparticular embodiments includes, but is not limited to:hairy/enhancer-of-split related with YRPW motif 1 (HEY1), UL16 bindingprotein 2 (ULBP2), hyaluronan synthase 1 (HAS1), GTP-binding protein GEM(GEM), renin (REN), collagen, type I, alpha 1 (COL1A1), cyclooxygenase 2(COX-2), angiopoietin 1 (ANGPT1), chemokine (C—X—C motif) ligand 6(CXCL6), prominin 1 (PROM1), bone morphogenetic protein 4 (BMP4),angiopoietin 2 (ANGPT2), inhibitor of kappaB kinase beta (IKBKB),platelet/endothelial cell adhesion molecule 1 (PECAM1), tyrosine kinasewith immunoglobulin-like and EGF-like domains 1 (TIE1), amphiregulin(AREG), caspase 3 (CASP3), jagged 1 (JAG1), aryl hydrocarbon receptornuclear translocator (ARNT), cAMP-responsive element modulator (CREM),connective tissue growth factor (CTGF), CD40 ligand (CD40L),BCL2-associated X protein (BAX), hepatocyte growth factor (HGF),superoxide dismutase 2 (SOD2), platelet derived growth factor B (PDGFB),thrombospondin 1 (THBS1), dual specificity protein phosphatase 4(DUSP4), cysteine-rich protein 61 (CYR61), chemokine (C—X—C motif)ligand 1 (CXCL1), endothelial tyrosine kinase (TEK), CASP8 and FADD-likeapoptosis regulator (CFLAR), insulin growth factor 2 (IGF2), chemokine(C—X—C motif) receptor 4 (CXCR4), matrix metalloprotease 2 (MMP2),fibroblast growth factor 2 (FGF2), prostaglandin-endoperoxide synthase 2(PTGS2), RAS-related C3 botulinum substrate 2 (RAC2), platelet derivedgrowth factor receptor (PDGFR), nuclear receptor subfamily 4, group A,member 2 (NR4A2), nuclear receptor subfamily 4, group A, member 3(NR4A3), telomerase reverse transcriptase (TERT), transforming growthfactor beta 1 (TGFB1), matrix metalloprotease 9 (MMP9), CD40 antigen(CD40), CD44 antigen (CD44), high mobility group box 1 (HMGB1), nitrogenoxide synthase 3 (NOS3), kinase insert domain receptor (KDR), integrinbeta 1 (ITGB1), catenin (cadherin-associated protein), beta 1 (CTNNB1),colony stimulating factor 3 (CSF3), interleukin 8 (IL8), plasminogenactivator, urokinase receptor (PLAUR), B-cell CLL/lymphoma 2 (BCL2),bone morphogenetic protein 2 (BMP2), colony stimulating factor 1 (CSF1),v-akt murine thymoma viral oncogene homolog 1 (AKT1), vascularendothelial growth factor A (VEGFA), intercellular adhesion molecule 1(ICAM1), chemokine (C—X—C motif) ligand 3 (CXCL3), caspase 8 (CASP8),CD34 antigen (CD34), interleukin 1A (ILIA), CD47 antigen (CD47),chemokine (C—C motif) ligand 7 (CCL7), hypoxia inducible factor 1A(HIF1A), EDN1 (endothelin 1), sphingosine-1-phosphate receptor 1(S1PR1), chemokine (C—C motif) receptor 1 (CCR1), SMAD family member 4(SMAD4), fms-related tyrosine kinase 1 (FLT1), CD151 antigen (CD151),placental growth factor (PGF), nuclear factor of kappa light polypeptidegene enhancer in B-cells 1 (NFKB1), SMAD family member 2 (SMAD2), CXCchemokine receptor 7 (CXCR7), transforming growth factor beta 3 (TGFB3),chemokine (C—X—C motif) ligand 5 (CXCL5), cyclin D1 (CCND1),heparin-binding EGF-like growth factor (HBEGF), nuclear receptorsubfamily 3, group C, member 1 (NR3C1), tumor necrosis factor (TNF),integrin alpha L (ITGAL), CXC chemokine receptor 2 (CXCR2), signaltransducer and activator of transcription 1 (STAT1), integrin alpha 4(ITGA4), leukemia inhibitory factor (LIF), RAS p21 protein activator 1(RASA1), cadherin 5 (CDHS), ephrin B2 (EFNB2), regulator of G-proteinsignaling 16 (RGS16), chemokine (C—X—C motif) ligand 2 (CXCL2), integrinalpha 5 (ITGA5), chemokine (C—X—C motif) ligand 12 (CXCL12), tissueinhibitor of metalloprotease 1 (TIMP1), Fos-related antigen 2 (FOSL2),integrin beta 2 (ITGB2), and tissue inhibitor of metalloprotease 2(TIMP2).

Another illustrative group of signature genes suitable for use inparticular embodiments includes, but is not limited to:hairy/enhancer-of-split related with YRPW motif 1 (HEY1), GTP-bindingprotein GEM (GEM), dual specificity protein phosphatase 4 (DUSP4),amphiregulin (AREG), Nuclear receptor related 1 protein (NR4A2), renin(REN), cAMP-responsive element modulator (CREM), collagen, type I, alpha1 (COL1A1), Fos-related antigen 2 (FOSL2), and UL16 binding protein 2(ULBP2).

Another illustrative group of signature genes suitable for use inparticular embodiments includes, but is not limited to: hyaluronansynthase 1 (HAS1), GTP-binding protein GEM (GEM), dual specificityprotein phosphatase 4 (DUSP4), amphiregulin (AREG), Nuclear receptorrelated 1 protein (NR4A2), renin (REN), cAMP-responsive elementmodulator (CREM), collagen, type I, alpha 1 (COL1A1), Fos-relatedantigen 2 (FOSL2), and CXC chemokine receptor 4 (CXCR1).

A further illustrative group of genes signature genes includes, but isnot limited to: CREM, GEM, NR4A2, NR4A3, ILIA, COX2, HEY1, CXCL2, CXCL3,and ULBP2.

Human HSPCs contacted with one or more agents and having enhancedtherapeutic properties further comprise increased levels ofintracellular cAMP signaling, e.g., CREB phosphorylation, or asdetermined by a biochemical assay; gene expression signatures indicatingupregulation of genes implicated in the PGE₂R₂/R4 cell signalingpathway, e.g., CREM, and genes that increase stem and progenitor cellhoming and engraftment, e.g., CXCR4, as determined by gene expressionassays, e.g., microarrays; no measurable decrease in stem and progenitorcell viability as determined by cell viability assays, e.g.,7-aminoactinomycinD (7-AAD) staining; and/or an increased capacity ofstem cells to self-renew as determined by an in vitro colony formingunits (CFU-C) assay, for example.

E. Agents

In various embodiments, compositions comprise blood cell products orhematopoietic stem or progenitor cells that have been contacted ortreated with one or more agents that enhance one or more therapeuticproperties of the cells. Without wishing to be bound to any particulartheory, it is contemplated that the compositions and methods disclosedelsewhere herein for preparing hematopoietic cells for transplantfurther comprises for short-term treatment of the hematopoietic cells.Hematopoietic cells frozen, thawed, and reconstituted or resuspended ina medium comprising a polysaccharide, and optionally HSA, stabilize cellviability such that they can be treated with one or more agents toincrease their therapeutic properties prior to transplant.

In one embodiment, a cryopreserved cell population is thawed andresuspended in a medium contemplated herein and then the hematopoieticcell population is modulated by contacting or culturing the cells withan agent. The cells may be thawed, resuspended, or modulated in the cellculture media of the invention containing a polysaccharide. As usedherein, “agent” refers to a compound or molecule capable of increasinggene expression of one or more genes that indicated an increase in atherapeutic property of the hematopoietic cells treated with the agent.Particular agents include, for example, compounds capable of stimulatingthe prostaglandin pathway, e.g., a cAMP analogue or enhancer, a Gα-sactivator, and a prostaglandin pathway agonist. Hematopoietic cells, inparticular embodiments, may be treated, cultured, or contacted with oneor more cytokines, growth factors, and/or glucocorticoids before and/orafter, or in addition to contacting the cells with one or more agentsthat stimulate the prostaglandin pathway or in lieu of contacting thehematopoietic cells with one or more agents that stimulate theprostaglandin pathway.

Hematopoietic cells may be treated under conditions sufficient toincrease the therapeutic properties of the cells. As used herein, theterms “conditions sufficient,” or “under conditions sufficient,” referto the conditions for treating the hematopoietic cells with one or moreagents to increase gene expression of one or more genes that indicatesthe increase in the therapeutic properties of the cells. Conditionsinclude, but are not limited to the source of the cells, the agents usedto treat the cells and concentrations of agent(s), the time the cellsare exposed to the agent(s), and the temperature of treatment.Therapeutic properties increased by contacting with one or more of theagents contemplated herein include, but are not limited to: engraftment,reconstitution, homing, survival, and proliferation.

In particular embodiments, compositions comprise one or more agents,each at a final concentration of about 1 μM to about 100 μM. In certainembodiments, compositions comprise one or more agents, each at a finalconcentration of about 1×10⁻¹⁴ M to about 1×10⁻³ M, about 1×10⁻¹³ M toabout 1×10⁻⁴ M, about 1×10⁻¹² M to about 1×10⁻⁵ M, about 1×10⁻¹¹ M toabout 1×10⁻⁴ M, about 1×10⁻¹¹ M to about 1×10⁻⁵ M, about 1×10⁻¹⁰ M toabout 1×10⁻⁴ M, about 1×10⁻¹⁰ M to about 1×10⁻⁵ M, about 1×10⁻⁹M toabout 1×10⁻⁴ M, about 1×10⁻⁹ M to about 1×10⁻⁵ M, about 1×10⁻⁸M to about1×10⁻⁴ M, about 1×10⁻⁷ M to about 1×10⁻⁴ M, about 1×10⁻⁶ M to about1×10⁻⁴ M, or any intervening ranges of final concentrations.

In another particular embodiment, compositions comprise blood cellproducts or hematopoietic cells treated with one or more agents, each ata final concentration of about 1×10⁻¹⁴ M, about 1×10⁻¹³ M, about1×10⁻¹²M, about 1×10⁻¹⁰ M, about 1×10⁻⁹ M, about 1×10⁻⁸ M, about 1×10⁻⁷M to about 1×10⁻⁶ M, about 1×10⁻⁵M, about 1×10⁻⁴ M, about 1×10⁻³ M, orany intervening final concentration. In treatments comprising one ormore agents, the agents can be at different concentrations from eachother or at the same concentration.

In particular embodiments, compositions comprise hematopoietic cellsthat are intermittently, episodically, or sequentially contacted withone or more agents within the same vessel (e.g., contacting thepopulation of cells with one drug for a period of time, exchanging theculture medium and/or washing the population of cells, then repeatingthe cycle with the same or a different combination of pharmaceuticalagents for the same predetermined period of time or a differentpredetermined period of time).

1. Prostaglandin Pathway Agonists

As used herein, the term “prostaglandin pathway agonist” refers to anagent that stimulates prostaglandin cell signaling pathways, includingan agent that stimulates the PGE₂R₂ and/or PGE₂R₄ cell signalingpathways, and increases CXCR4 gene expression in the cells. Illustrativeexamples of prostaglandin pathway agonists that are suitable for use inpreparing cells of the invention, include, but are not limited to, PGE₂,dmPGE₂, 15(S)-15-methyl PGE₂, 20-ethyl PGE₂,8-iso-16-cyclohexyl-tetranor PGE₂, and PGE₂ analogues. In certainembodiments, PGE₂R₂ and PGE₂R₄ agonists and analogues thereof are ofparticular interest, and in some embodiments, the agent preferentiallybinds and activates a PGE₂EP₂ or PGE₂EP₄ receptor.

As used herein, the terms “prostaglandin E₂” or “PGE₂” include, withoutlimitation, any naturally-occurring or chemically synthesized PGE2molecule, as well as “analogues” thereof. As used herein, the term“analogue” or relates to a chemical molecule that is similar to anotherchemical substance, e.g., PGE₂, in structure and function, oftendiffering structurally by a single element or group, but may differ bymodification of more than one group (e.g., 2, 3, or 4 groups) if itretains the same function as the parental chemical. Such modificationsare routine to persons skilled in the art, and include, for example,additional or substituted chemical moieties, such as esters or amides ofan acid, protecting groups such as a benzyl group for an alcohol orthiol, and tert-butoxylcarbonyl groups for an amine. Also included aremodifications to alkyl side chains, such as alkyl substitutions (e.g.,methyl, dimethyl, ethyl, etc.), modifications to the level of saturationor unsaturation of side chains, and the addition of modified groups suchas substituted phenyl and phenoxy. Analogues can also includeconjugates, such as biotin or avidin moieties, enzymes such ashorseradish peroxidase and the like, and including radio-labeled,bioluminescent, chemoluminescent, or fluorescent moieties. Also,moieties may be added to the agents described herein to alter theirpharmacokinetic properties, such as to increase half-life in vivo or exvivo, or to increase their cell penetration properties, among otherdesirable properties. Also included are prodrugs, which are known toenhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.) (see, e.g.,WO/2006/047476 for exemplary EP agonist prodrugs, which is incorporatedby reference for its disclosure of such agonists).

Illustrative examples of PGE₂ “analogues” include, without limitation,16,16-dimethyl PGE₂ (“dmPGE₂”), 16,16-dimethyl PGE₂p-(p-acetamidobenzamido) phenyl ester, 11-deoxy-16,16-dimethyl PGE₂,9-deoxy-9-methylene-16, 16-dimethyl PGE₂, 9-deoxy-9-methylene PGE₂,9-keto Fluprostenol, 5-trans PGE₂, 17-phenyl-omega-trinor PGE₂, PGE₂serinol amide, PGE₂ methyl ester, 16-phenyl tetranor PGE₂,15(S)-15-methyl PGE₂, 15(R)-15-methyl PGE₂, 8-iso-15-keto PGE₂, 8-isoPGE2 isopropyl ester, 8-iso-16-cyclohexyl-tetranor PGE₂, 20-hydroxyPGE₂, 20-ethyl PGE₂, 11-deoxy PGE₁, nocloprost, sulprostone, butaprost,15-keto PGE₂, and 19 (R) hydroxy PGE₂. Also included are PG analogues orderivatives having a similar structure to PGE₂ that are substituted withhalogen at the 9-position (see, e.g., WO 2001/12596, herein incorporatedby reference in its entirety), as well as 2-decarboxy-2-phosphinicoprostaglandin derivatives, such as those described in U.S. PublicationNo. 2006/0247214, herein incorporated by reference in its entirety).

Stimulation/activation of the PGE₂R₂ (EP₂) and PGE₂R₄ (EP₄) cellsignaling pathways are contemplated to underlie the physiologicalresponses in HSPCs that increase engraftment, maintain cell viability,and increase homing and proliferation of the cells. Accordingly, in oneembodiment, a “non-PGE₂-based ligand” that binds to and stimulatesPGE₂R₂ and PGE₂R₄ receptors (i.e., a PGE₂R₂/PGE₂R₄ agonist) iscontemplated for use in the methods of the invention.

Illustrative examples of non-PGE₂-based EP₂ receptor agonists includeCAY10399, ONO_8815Ly, ONO-AE1-259, CP-533,536 and carbazoles andfluorenes disclosed in WO 2007/071456.

Illustrative examples of non-PGE₂-based EP₄ agonists include ONO-4819,APS-999 Na, AH23848, ONO-AE1-329, and other non-PGE₂-based EP₄ agonistsdisclosed in WO/2000/038663; U.S. Patent No. 6,747,037; and U.S. PatentNo. 6,610,719).

Agents selective for the PGE₂ EP₄ receptor preferentially bind to andactivate PGE₂ EP₄ receptors. Such agents have a higher affinity for theEP₄ receptor than for any of the other three EP receptors namely EP₁,EP₂ and EP₃. Agents that selectively bind the PGE EP₄ receptor include,but are not limited to, agents selected from the group consisting of:5-[(1E,3R)-4,4-difluoro-3-hydroxy-4-phenyl-1-buten-1-yl]-1-[6-(2H-tetrazol-5R-yl)hexyl]-2-pyrrolidinone;2-[3-[(1R,2S,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-5-[2-(methoxymethyl)phenyl]pent-1-enyl]-5-oxocyclopentyljsulfanylpropylsulfanyl]aceticacid; methyl 4-[2-(1R,2R,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-4-[3-(methoxymethyl)phenyl]but-1-enyl]-5-oxocyclopentyl]ethylsulfanyl]butanoate;16-(3-Methoxymethyl)phenyl-ro-tetranor-5-thiaPGE;5-{3-(2S)-2-{(3R)-3-hydroxy-4-[3-(trifluoromethyl)phenyl]butyl}-5-oxopyrrolidin-1-yl]propyl]thiophene-2-carboxylate;[4′-[3-butyl-5-oxo-1-(2-trifluoromethyl-phenyl)-1,5-dihydro-[1,2,4]triazol-4-ylmethyl]-biphenyl-2-sulfonicacid (3-methyl-thiophene-2-carbonyl)-amide]; and((Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-enoicacid), and pharmaceutically acceptable salts of any of these agents.

In particular embodiments, the prostaglandin pathway agonist is PGE₂,dmPGE₂, 15(S)-15-methyl PGE₂, 20-ethyl PGE₂, or8-iso-16-cyclohexyl-tetranor PGE₂.

2. cAMP Enhancer

A “cyclic AMP (cAMP) enhancer,” refers to a molecule that produces orcauses a greater amount of cAMP in a cell, or a greater amount of cAMPactivity in a cell, or any other relevant component of a cAMP relatedsignal transduction pathway, or a measurable downstream physiologicalresponse or effect of a cAMP signaling pathway, as compared to no agentor a control molecule/composition.

Illustrative examples of cAMP enhancers include, but are not limited tophorbol ester, forskolin, sclareline, 8-bromo-cAMP, cholera toxin (CTx),aminophylline, 2,4 dinitrophenol (DNP), norepinephrine, epinephrine,isoproterenol, isobutylmethylxanthine (IBMX), caffeine, theophylline(dimethylxanthine), dopamine, rolipram, iloprost, prostaglandin E1,prostaglandin E2, pituitary adenylate cyclase activating polypeptide(PACAP), and vasoactive intestinal polypeptide (VIP), among others knownin the art. Other examples of cAMP enhancers include cAMP and analoguesof cAMP, such sp-5,6-DC1-BIMPS (BIMPS) and dibutyryl cAMP (dbcAMP),among others.

3. Gα-s Activators

A “Gα-s activator or activating agent” or “G-protein alpha-s activatoror activating agent” includes any molecule capable of activating thealpha subunit of the stimulatory G-protein (“Gα-s”) or variants of Gα-s.

Illustrative examples of Gα-s activators include PGE₂ and agonists andderivatives thereof, and cholera toxin.

4. Glucocorticoids

Illustrative examples of glucocorticoids and glucocorticoid receptoragonists suitable for use in the methods of the invention include, butare not limited to, medrysone, alclometasone, alclometasonedipropionate, amcinonide, beclometasone, beclomethasone dipropionate,betamethasone, betamethasone benzoate, betamethasone valerate,budesonide, ciclesonide, clobetasol, clobetasol butyrate, clobetasolpropionate, clobetasone, clocortolone, cloprednol, cortisol, cortisone,cortivazol, deflazacort, desonide, desoximetasone, desoxycortone,desoxymethasone, dexamethasone, diflorasone, diflorasone diacetate,diflucortolone, diflucortolone valerate, difluorocortolone,difluprednate, fluclorolone, fluclorolone acetonide, fludroxycortide,flumetasone, flumethasone, flumethasone pivalate, flunisolide,flunisolide hemihydrate, fluocinolone, fluocinolone acetonide,fluocinonide, fluocortin, fluocoritin butyl, fluocortolone,fluorocortisone, fluorometholone, fluperolone, fluprednidene,fluprednidene acetate, fluprednisolone, fluticasone, fluticasonepropionate, formocortal, halcinonide, halometasone, hydrocortisone,hydrocortisone acetate, hydrocortisone aceponate, hydrocortisonebuteprate, hydrocortisone butyrate, loteprednol, meprednisone,6a-methylprednisolone, methylprednisolone, methylprednisolone acetate,methylprednisolone aceponate, mometasone, mometasone furoate, mometasonefuroate monohydrate, paramethasone, prednicarbate, prednisolone,prednisone, prednylidene, rimexolone, tixocortol, triamcinolone,triamcinolone acetonide and ulobetasol, as well as combinations thereof.

In particular embodiments, the glucocorticoid comprises medrysone,hydrocortisone, triamcinolone, alclometasone, or dexamethasone. In moreparticular embodiments, the glucocorticoid is medrysone.

F. Hematopoietic Cell Preparation Methods

Methods contemplated herein provide improved preparation of blood cellproducts and/or hematopoietic cells for transplants. In particularembodiments, a method comprises either or both of cryopreservation andthawing of blood cell products or hematopoietic cells and transfer ofthe thawed cells into a culture medium contemplated herein.Hematopoietic cells may be isolated, modulated, and/or expanded eitherprior to cryopreservation or following thawing of a previouslycryopreserved or noncryopreserved (fresh) blood cell product. Withoutwishing to be bound by any particular theory, the culture media iscontemplated to be useful in resuspension, processing, isolating,modulating, and/or expanding fresh or frozen blood cells products. Inone embodiment, the culture media improve cell viability, decrease celllysis, and increase the biological activity and therapeutic propertiesof fresh blood cell products. Accordingly, cryopreserved or thawed cellsor fresh cells can further be isolated, modulated, or expanded in aculture medium contemplated herein.

In one embodiment, a method of stabilizing a hematopoietic cellpopulation for transplantation is provided. In a particular embodiment,the method comprises thawing a cryopreserved blood cell product orpopulation of hematopoietic cells and transfer into a culture mediumcontemplated herein. The stabilized cell population has reduced celllysis and increased CD34⁺ cell viability compared to a thawed controlcell population that has been transferred to a control solution, e.g.,10% dextran, 5% HSA, and 0.9% NaCl, or defined culture medium alone.

In a particular embodiment, a method of reducing hematopoietic celllysis in a population of cells for transplantation is provided. In aparticular embodiment, the method comprises thawing a cryopreservedblood cell product or population of hematopoietic cells and transferinto a culture medium contemplated herein. In particular embodiments,the lysis is reduced about 10%, about 20%, about 30%, about 40%, about50%, about two-fold, about three-fold, or about five-fold compared to athawed control cell population that has been transferred to a controlsolution.

In one embodiment, a method of increasing hematopoietic cell viabilityin a population of cells for transplantation is provided. In aparticular embodiment, the method comprises thawing a cryopreservedblood cell product or population of hematopoietic cells and transfer itinto a culture medium contemplated herein. In certain embodiments, theCD34⁺ cell viability is increased about 10%, about 20%, about 30%, about40%, about 50%, about two-fold, about three-fold, or about five-foldcompared to a thawed control cell population that has been transferredto a control solution.

In a certain embodiment, a method of increasing total nucleated cell(TNC) count in a population of cells for transplantation is provided. Ina particular embodiment, the method comprises thawing a cryopreservedblood cell product or population of hematopoietic cells and transferringinto a culture medium contemplated herein. In certain embodiments, theTNC is increased about 10%, about 20%, about 30%, about 40%, about 50%,about two-fold, about three-fold, or about five-fold compared to athawed control cell population that has been transferred to a controlsolution.

1. Cryopreservation

In one embodiment, a blood cell product or population of hematopoieticcells can be divided and frozen in one or more bags (or units). Inanother embodiment, two or more cell populations can be pooled, dividedinto separate aliquots, and each aliquot frozen. As used herein, theterms “frozen/freezing” and “cryopreserved/cryopreserving” are usedinterchangeably. In particular embodiments, cryopreservation includesknown methods that freeze cells in viable form. Cryopreservation causescell injury by osmotic effects on the cell membrane, cell dehydration,solute concentration, and ice crystal formation. As ice forms outsidethe cell, available water is removed from solution and withdrawn fromthe cell, causing osmotic dehydration and raised solute concentrationwhich may eventually destroy the cell. For a discussion, see Mazur, P.,1977, Cryobiology 14:251-272.

These injurious effects can be reduced by using a cryoprotective agentsuch as dimethyl sulfoxide (DMSO), glycerol, polyvinylpyrrolidine,polyethylene glycol, ethylene glycol, i-erythritol, D-ribitol,D-mannitol, D-sorbitol, i-inositol, D-lactose, choline chloride, aminoacids, methanol, acetamide, glycerol monoacetate, and inorganic salts,(b) control of the freezing rate, and (c) storage at a temperaturesufficiently low to minimize degradative reactions. In a preferredembodiment, DMSO is used, a liquid which is nontoxic to cells in lowconcentration. Being a small molecule, DMSO freely permeates the celland protects intracellular organelles by combining with water to modifyits freezability and prevent damage from ice formation. Addition ofplasma (e.g., to a concentration of 20-25%) or a plasma substitute(Plasmalyte) can augment the protective effect of DMSO. After additionof DMSO, cells should be kept at 0° C. until freezing, since DMSOconcentrations of about 1% are toxic at temperatures above 4° C.

A controlled slow cooling rate can be used to reduce freezing-inducedcellular damage. Different cryoprotective agents have different optimalcooling rates. The cooling procedure can be carried out by use of, e.g.,a programmable freezing device or a methanol bath procedure.

After thorough freezing, the blood cell product or hematopoietic cellscan be rapidly transferred to a long-term cryogenic storage vessel. Inone embodiment, samples can be cryogenically stored in liquid nitrogen(−196° C.) or its vapor (−165° C.). Such storage is greatly facilitatedby the availability of highly efficient liquid nitrogen refrigerators,which resemble large Thermos containers with an extremely low vacuum andinternal super insulation, such that heat leakage and nitrogen lossesare kept to an absolute minimum. Suitable racking systems arecommercially available and can be used for cataloguing, storage, andretrieval of individual specimens.

2. Thawing

In various embodiments, compositions and methods contemplated hereinoffer numerous advantages in the thawing of previously frozen blood cellproducts and hematopoietic cells. Without wishing to be bound by anyparticular theory, it is contemplated that thawing frozen blood cellproducts and hematopoietic cells and transferring the thawed cells intothe culture media, contemplated herein increases total nuclear cell(TNC) count and hematopoietic cell viability and allows for subsequentprocessing, modulation, and/or expansion of the cells. It is furthercontemplated that thawing blood cell products, and transferring thethawed cells into the culture media stabilizes the membranes ofapoptosing cells, thereby decreasing cell lysis, preventing cell debrisaggregation, and preventing the release of intracellular components thatcan inhibit cellular processes; thus, the method of thawing blood cellproducts and transferring the thawed cells into in the culture mediacontemplated herein provides populations of higher quality hematopoieticcells for transplant therapies.

In one embodiment, a cryopreserved population of cells comprising DMSOor other cryoprotective agents is thawed and diluted about two-fold,about three-fold, about four-fold, about five-fold, about six-fold,about seven-fold, about eight-fold, about nine-fold, about ten-fold ormore in a culture medium contemplated herein and used in subsequentsteps of preparing the cells for transplant. The diluted cellcomposition can then be washed and resuspended in a culture mediumcontemplated herein or used in subsequent steps of preparing the cellsfor transplant. In a particular embodiment, the thawed cells can then befurther processed, modulated, or expanded. In a certain embodiment, thethawed cells can be directly infused into a human subject in needthereof.

In one embodiment, a method of preparing cryopreserved blood cellproducts for transplantation is provided. In particular embodiments, themethod comprises thawing a cryopreserved blood cell product orpopulation of hematopoietic cells, e.g., hematopoietic stem andprogenitor cells. In other embodiments, the thawed cells are transferredinto a culture medium contemplated herein, and optionally prepared forinfusion into a subject, either immediately or following one or moresteps of processing, modulation, or expansion.

In a particular embodiment, the blood cell product or hematopoieticcells are thawed at a temperature of about 20° C. to about 37° C., about25° C. to about 37° C., about 30° C. to about 37°, or about 35° C. toabout 37° C. In a certain embodiment, the blood cell product orhematopoietic cells are thawed at a temperature of about 20° C., about21° C., about 22° C., about 23° C., about 24° C., about 25° C., about26° C., about 27° C., about 28° C., about 29° C., about 30° C., about31° C., about 32° C., about 33° C., about 34° C., about 35° C., about36° C., or about 37° C.

3. Processing Hematopoietic Cells

In various embodiments, methods contemplated herein provide improvedpreparation of blood cell products and/or hematopoietic cells fortransplants comprising a step of processing the blood cell products orcells. As used herein, the term “processing” refers to a step ofwashing, purifying, or otherwise manipulating a blood cell product.

Blood cell products can be washed any number of times between otherisolation, freezing, thawing, resuspending, modulating, and expandingsteps. For example, blood cell products may be washed 1, 2, 3, 4, 5 ormore times between each step of manipulating or handling the blood cellproduct. Illustrative wash solutions for washing blood cell productsinclude physiological saline, Ringer's solution, low molecular weightdextran, and HSA in 0.9% NaCl. In particular embodiments, a blood cellproduct may also be washed one or more times with a chemically definedmedium.

In other embodiments, a blood cell product may be processed so as toremove portions of the product to improve downstream manipulation. Forexample, whole cord blood, placental blood, or mobilized peripheralblood may be processed to remove red blood cells and plasma. Inparticular embodiments, red blood cells are removed to minimize bloodtype incompatibilities reactions to between donor and recipient.

In one embodiment, the blood cell product or hematopoietic cells areprocessed such that the cell population is about 75%, about 80%, about85%, about 90%, about 95%, or about 100% hematopoietic cells or CD34⁺cells. In some embodiments, the population of cells is less than about0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, or 30% hematopoietic cellsor CD34⁺ cells.

In particular embodiments, the processed population of cells comprisespopulations of hematopoietic stem or progenitor cells or CD34⁺ cells andis substantially free of mesenchymal stem cells and/or endothelialprogenitor cells. In certain embodiments, the population of cellscomprises hematopoietic stem or progenitor cells or CD34⁺ cells lessthan about 30%, 25%, 20%, 15%, 10% or 5% mesenchymal stem cells and lessthan about 30%, 25%, 20%, 15%, 10% or 5% endothelial progenitor cells.

Populations of cells may alternatively be depleted of mesenchymal stemcells and/or endothelial progenitor cells using methods known in theart, for example, using immunomagnetic selection techniques,fluorescence activated cell sorting, or a combination therein, CD34⁺cells may be purified from any number of cell sources disclosed hereinand suitable for use in the present invention.

4. Modulating Hematopoietic Cells

In various embodiments, use of the culture media contemplated hereinoffers particular advantages over existing methods of manipulating bloodcell products. In addition to increasing cell viability, the culturemedia allow manipulated cells to retain or increase biological activityand therapeutic properties. Products such as whole cord blood that iscultured in the culture media contemplated herein comprise changes ingene expression, including increases in gene expression that arerepresentative of a therapeutic gene expression signature, that arenormally low or absent in the same products treated in the absence ofthe culture media contemplated herein. For example, whole cord bloodtreated with one or more agents (e.g., a prostaglandin pathway agonist)as described elsewhere herein shows increased gene expression ofsignature genes that indicate that the cells are imbued with therapeuticproperties compared to whole cord blood manipulated in controlsolutions, as described elsewhere herein.

In one embodiment, compositions comprise blood cell products orhematopoietic cells treated ex vivo with one or more agents capable ofincreasing CXCR4 gene expression under conditions sufficient to increaseCXCR4 gene expression at least about 2, 3, 4, 5, 10, 15, 20, 30, 40, 50,60, 70, or 80 fold in the contacted cells compared to non-contactedcells. In particular embodiments, the hematopoietic cells are contactedwith one or more agents after thawing a frozen blood cell product orhematopoietic cells from a subject. In another embodiment, hematopoieticcells are cryopreserved; thawed, resuspended, and/or purified in aculture medium contemplated herein, and modulated and/or expanded bycontacting the cells with one or more agents in a culture mediumcontemplated herein.

In particular embodiments, HSPCs are treated with one or more agents inan amount effective and for a time sufficient (i.e., under conditionssufficient) to increase CXCR4 gene expression at least about 2, 3, 4, 5,10, 15, 20, 30, 40, 50, 60, 70, or 80 fold in the contacted cellscompared to non-contacted cells.

In certain embodiments, sufficient temperature conditions includeincubation of the blood cell product or hematopoietic cells with the oneor more agents at a physiologically relevant temperature, such as atemperature range of about 22° C. to about 39° C. (about roomtemperature to about body temperature), including but not limited totemperatures of about 22° C., 23° C., 24° C., 25° C., 26° C., 27° C.,28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C.,37° C., 38° C., and 39° C. In a particular embodiment, the sufficienttemperature condition is between about 35° C. and 39° C. In oneembodiment, the sufficient temperature condition is about 37° C. In aparticular embodiment, a sufficient concentration of an agent is a finalconcentration of about 10 nM to about 100 μM, about 100 nM, about 500nM, about 1 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM,about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about100 μM, about 110 μM, or about 120 μM, or any other interveningconcentration of the agent (e.g., 0.1 μM, 1 μM, 5 μM, 10 μM, 20 μM, 50μM, 100 μM). In a particular embodiment, the sufficient concentration ofeach agent is a final concentration of about 10 μM to about 25 μM. Inone embodiment, the sufficient concentration of an agent is a finalconcentration of about 10 μM.

In further embodiments, the sufficient time period for treating a bloodcell product or a population of hematopoietic cells with one or moreagents is an incubation period of about 60 minutes to about 24 hours,about 60 minutes to about twelve hours, about 60 minutes to about 6hours, about 2 hours to about 6 hours, about 2 hours to about 4 hours,and including, but not limited to, treatment for a duration of about 60minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100minutes, about 110 minutes, about 2 hours, about 2.5 hours, about 3hours, about 3.5 hours or about 4 hours or any other interveningduration. In a particular embodiment, the sufficient incubation periodis about 2 hours to about 4 hours. In one embodiment, the sufficientincubation period for treating the HSPCs is about four hours.

In various embodiments, conditions sufficient to increase CXCR4 geneexpression at least about 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, or80 fold in the contacted cells compared to non-contacted cells comprisestreating HSPCs ex vivo at a temperature range of about 22° C. to about39° C.; at a final concentration of about 10 μM to about 25 μM of aprostaglandin pathway agonist, and about 10 μM to about 25 μM of aglucocorticoid; and incubation with the agents for about 1 hour to about4 hours, for about 2 hours to about 3 hours, for about 2 hours to about4 hours, or for about 3 hours to about 4 hours.

In particular embodiments, conditions sufficient to increase CXCR4 geneexpression at least about 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, or80 fold in the contacted cells compared to non-contacted cells comprisestreating HSPCs ex vivo at a temperature range of about 22° C. to about39° C.; at a final concentration of about 10 μM to about 25 μM of PGE₂or dmPGE₂, and about 10 μM to about 25 μM of a glucocorticoid; andincubation with the agents for about 1 hour to about 4 hours, for about2 hours to about 3 hours, for about 2 hours to about 4 hours, or forabout 3 hours to about 4 hours.

In various embodiments, conditions sufficient to increase CXCR4 geneexpression at least about 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, or80 fold in the contacted cells compared to non-contacted cells comprisestreating HSPCs ex vivo at a temperature range of about 37° C., at afinal concentration of about 10 μM 16,16-dmPGE₂, for a duration of about2 hours.

In particular embodiments, methods contemplated herein comprisemodulating a blood cell product or hematopoietic cells to increase geneexpression of two or more genes in a gene expression signature or panel.

One illustrative example of a suitable gene expression panel includesHEY1, COX2, ULBP2, HAS1, GEM1, REN, COL1A1, ANGPT1, CXCL6, PROM1, BMP4,ANGPT2, IKBKB, PECAM1, TIE1, AREG, CASP3, JAG1, ARNT, CREM, CTGF, CD40L,BAX, HGF, SOD2, PDGFB, THBS1, DUSP4, CYR61, CXCL1, TEK, CFLAR, IGF2,CXCR4, MMP2, FGF2, PTGS2, RAC2, PDGFR, NR4A2, NR4A3, TERT, TGFB1, MMP9,CD40, CD44, HMGB1, NOS3, KDR, ITGB1, CTNNB1, CSF3, IL8, PLAUR, BCL2,BMP2, CSF1, AKT1, VEGFA, ICAM1, CXCL3, CASP8, CD34, ILIA, CD47, CCL7,HIF1A, EDN1, S1PR1, CCR1, SMAD4, FLT1, CD151, PGF, NFKB1, SMAD2, CXCR7,TGFB3, CXCL5, CCND1, HBEGF, NR3C1, TNF, ITGAL, CXCR2, STAT1, ITGA4, LIF,RASA1, CDH5, EFNB2, RGS16, CXCL2, ITGA5, CXCL12, TIMP1, FOSL2, ITGB2,and TIMP2.

In another embodiment, a blood cell product or population ofhematopoietic cells is modulated to increase gene expression of aplurality of the signature genes selected from the group consisting of:CREM, GEM, NR4A2, NR4A3, ILIA, COX2, HEY1, CXCL2, CXCL3, and ULBP2. Themodulated cells may have increased expression of 2, 3, 4, 5, 6, 7, 8, 9,or 10 signature genes compared to expression levels in control oruntreated cells.

In particular embodiments, a blood cell product or population ofhematopoietic cells is modulated to increase gene expression of at leasttwo genes, at least five genes, at least 10 genes, at least 25 genes, atleast 50 genes, or at least 100 or more genes, or any intervening numberof signature genes. In preferred embodiments, a blood cell product orpopulation of hematopoietic cells is modulated to increase geneexpression of about 2 to about 25 genes, about 2 to about 10 genes, orabout 5 to about 10 genes, or any intervening range of genes thereof.

In certain embodiments, a blood cell product or population ofhematopoietic cells is deemed to be sufficiently modulated when theexpression of at least 2, 3, 4, or 5 signature genes is increased about80-fold, about 70-fold, about 60-fold, about 50-fold, about 40-fold,about 30-fold, about 20-fold, about 10-fold, about 5-fold, about 3-fold,or about 2-fold compared to expression of the genes in a controlpopulation of cells. In additional embodiments, a blood cell product orpopulation of hematopoietic cells is sufficiently modulated when theexpression of at least 10, 20, 30, 40, 50 ,60, 70, 80, or 90 signaturegenes, or any intervening number of genes thereof, is increased about80-fold, about 70-fold, about 60-fold, about 50-fold, about 40-fold,about 30-fold, about 20-fold, about 10-fold, about 5-fold, about 3-fold,or about 2-fold compared to expression of the genes in a controlpopulation cells.

In various embodiments, the modulated hematopoietic cells compriseincreased therapeutic properties including, increased engraftment,increased engraftment potential, increased hematopoietic reconstitution,increased hematopoietic reconstitution potential, increased homing,increased homing potential, increased proliferation and increasedproliferation potential. In particular embodiments, these increasedtherapeutic properties are increased compared to a blood cell product orhematopoietic cells contacted with a control or vehicle composition (10%dextran, 5% HSA, 0.9% NaCl or defined culture medium alone).

5. Expanding Hematopoietic Cells

In one embodiment, compositions comprise blood cell products or apopulation of cells comprising hematopoietic cells treated ex vivo withone or more agents to expand hematopoietic stem and progenitor cells. Ina particular embodiment, cells are contacted with one or more agentsthat promote growth and expansion of hematopoietic stem and progenitorcells during ex vivo treatments, such as prior to, during, and/or aftertransplant procedures (see, e.g., WO 2008/073748, herein incorporated byreference in its entirety). Likewise, also according to the methodsprovided herein, ex vivo expansion of HSPCs in the presence of aprostaglandin pathway agonist or an analog thereof prior tohematopoietic cell transplantation can improve engraftment andreconstitution of hematopoiesis and immune function after transplant(see, e.g., Lord et al., Cell Cycle 6:3054-7, 2007, herein incorporatedby reference in its entirety).

Therefore, in particular embodiments, methods of preparing cells for atransplant contemplated herein comprise stimulating hematopoietic stemcell (HSC) growth or expansion, as well as the growth or expansion ofother stem-like cells (e.g., multi-potent cells, pluripotent cells,etc.) comprising transferring the blood cell product or hematopoieticcell population into a culture medium contemplated herein, contactingthe cells with one or more prostaglandin pathway agonists, andincubating the cells for a time sufficient to stimulate growth orexpansion of the HSPCs, and thereby stimulating HPSC growth orexpansion.

G. Methods of Treatment or Therapeutic Methods

The compositions and methods of preparing cells contemplated herein areuseful in a variety of clinical settings, including celltransplantation, treatment of hematological disorders, diseases, andconditions, treatment of ischemia, and gene therapy. In particularembodiments, the compositions comprising blood cell products orhematopoietic cells are useful in increasing engraftment,reconstitution, homing, and proliferation of cell grafts in a subject inneed thereof.

“Subjects in need thereof” include, but are not limited to a subject inneed of hematopoietic engraftment, reconstitution, homing,proliferation, or gene therapy. Included are subjects that have or thathave been diagnosed with various types of leukemias, anemias, lymphomas,myelomas, immune deficiency disorders, and solid tumors as discussedelsewhere herein. A “subject” also includes a human who is a candidatefor stem cell transplant or bone marrow transplantation, such as duringthe course of treatment for a malignant disease or a component of genetherapy. In particular embodiments, a subject receives geneticallymodified HSPCs as a cell-based gene therapy. Subjects may also includeindividuals or animals that donate stem cells or bone marrow forallogeneic transplantation. In certain embodiments, a subject may haveundergone myeloablative irradiation therapy or chemotherapy, or may haveexperienced an acute radiation or chemical insult resulting inmyeloablation. In certain embodiments, a subject may have undergoneirradiation therapy or chemotherapy, such as during various cancertreatments. Typical subjects include animals that exhibit aberrantamounts (lower or higher amounts than a “normal” or “healthy” subject)of one or more physiological activities that can be modulated by anagent or a stem cell or marrow transplant.

Subjects in need of hematopoietic engraftment or reconstitution includesubjects undergoing chemotherapy or radiation therapy for cancer, aswell as subjects suffering from (e.g., afflicted with) non malignantblood disorders, particularly immunodeficiencies (e.g. SCID, Fanconi'sanemia, severe aplastic anemia, or congenital hemoglobinopathies, ormetabolic storage diseases, such as Hurler's disease, Hunter's disease,mannosidosis, among others) or cancer, particularly hematologicalmalignancies, such as acute leukemia, chronic leukemia (myeloid orlymphoid), lymphoma (Hodgkin's or non-Hodgkin's), multiple myeloma,myelodysplastic syndrome, or non-hematological cancers such as solidtumors (including breast cancer, ovarian cancer, brain cancer, prostatecancer, lung cancer, colon cancer, skin cancer, liver cancer, orpancreatic cancer). Subjects also include subjects suffering fromaplastic anemia, an immune disorder (severe combined immune deficiencysyndrome or lupus), myelodysplasia, thalassemaia, sickle-cell disease orWiskott-Aldrich syndrome. In some embodiments, the subject suffers froma disorder that is the result of an undesired side effect orcomplication of another primary treatment, such as radiation therapy,chemotherapy, or treatment with a bone marrow suppressive drug, such aszidovadine, chloramphenical or gangciclovir. Such disorders includeneutropenias, anemias, thrombocytopenia, and immune dysfunction.

Other subjects may have disorders caused by an infection (e.g., viralinfection, bacterial infection or fungal infection) which causes damageto stem or progenitor cells of the bone marrow.

In addition, subject suffering from the following conditions can alsobenefit from treatment using cell-based compositions of the invention:lymphocytopenia, lymphorrhea, lymphostasis, erythrocytopenia,erthrodegenerative disorders, erythroblastopenia, leukoerythroblastosis;erythroclasis, thalassemia, myelofibrosis, thrombocytopenia,disseminated intravascular coagulation (DIC), immune (autoimmune)thrombocytopenic purpura (ITP), HIV inducted ITP, myelodysplasia;thrombocytotic disease, thrombocytosis, congenital neutropenias (such asKostmann's syndrome and Schwachman-Diamond syndrome), neoplasticassociated-neutropenias, childhood and adult cyclic neutropaenia;post-infective neutropaenia; myelo-dysplastic syndrome; neutropaeniaassociated with chemotherapy and radiotherapy; chronic granulomatousdisease; mucopolysaccharidoses; Diamond Blackfan; sickle cell disease;β-thalassemia major; Gaucher's disease; Krabbe's disease; metachromaticleukodystrophy; Tay-Sachs; Nieman Pick; glycoproteinoses (e.g.,fucosidosis, a-mannosidosis); and MPS-III (Sanfillipo).

In a particular embodiment, the subject is a bone marrow donor who hasdonated bone marrow, is a bone marrow donor who has yet to donate bonemarrow, is a bone marrow donor transplant recipient, has hematopoieticprogenitor cells under environmental stress, has anemia, has a reducedlevel of immune cell function compared to a normal subject, or has animmune system deficiency.

In a certain embodiment, the subject has myeloma, non-Hodgkin'slymphoma, Hodgkin's lymphoma, chronic myeloid leukemia, chronicmyelogenous leukemia, chronic granulocytic leukemia, acute lymphoblasticleukemia, acute nonlymphoblastic leukemia, or pre-leukemia.

Subject also include those in need of treatment for ischemic tissue orone or more symptoms associated with tissue ischemia, including, but notlimited to, impaired, or loss of, organ function (including withoutlimitation impairments or loss of brain, kidney, or heart function),cramping, claudication, numbness, tingling, weakness, pain, reducedwound healing, inflammation, skin discoloration, and gangrene. As usedherein, the terms “ischemia,” “ischemic condition,” or “ischemic event”mean any decrease or stoppage in the blood supply to any cell, tissue,organ, or body part caused by any constriction, damage, or obstructionof the vasculature. Ischemia sometimes results from vasoconstriction orthrombosis or embolism. Ischemia can lead to direct ischemic injury,tissue damage due to cell death caused by reduced supply of oxygen(hypoxia, anoxia), glucose, and nutrients. “Hypoxia” or a “hypoxiccondition” intends a condition under which a cell, organ or tissuereceives an inadequate supply of oxygen. “Anoxia” refers to a virtuallycomplete absence of oxygen in the organ or tissue, which, if prolonged,may result in death of the cell, organ or tissue.

In particular embodiments, the subject is in need of gene therapy, suchas, for example, a hemoglobinopathy. As used herein, the term“hemoglobinopathy” or “hemoglobinopathic condition” includes anydisorder involving the presence of an abnormal hemoglobin molecule inthe blood. Examples of hemoglobinopathies included, but are not limitedto, hemoglobin C disease, hemoglobin sickle cell disease (SCD), sicklecell anemia, and thalassemias. Also included are hemoglobinopathies inwhich a combination of abnormal hemoglobins is present in the blood(e.g., sickle cell/Hb-C disease).

The term “sickle cell anemia” or “sickle cell disease” is defined hereinto include any symptomatic anemic condition which results from sicklingof red blood cells. Manifestations of sickle cell disease include:anemia; pain; and/or organ dysfunction, such as renal failure,retinopathy, acute-chest syndrome, ischemia, priapism and stroke. Asused herein the term “sickle cell disease” refers to a variety ofclinical problems attendant upon sickle cell anemia, especially in thosesubjects who are homozygotes for the sickle cell substitution in HbS. Asused herein, the term “thalassemia” encompasses hereditary anemias thatoccur due to mutations affecting the synthesis of hemoglobin. Thus, theterm includes any symptomatic anemia resulting from thalassemicconditions such as severe or β-thalassemia, thalassemia major,thalassemia intermedia, α-thalassemias such as hemoglobin H disease.

In one embodiment, a method of cell-based therapy, comprisesadministering to a subject in need thereof, a blood cell product or apopulation of hematopoietic cells thawed and transferring into a culturemedium contemplated herein, optionally wherein the cells have beenprocessed, modulated, or expanded. In various embodiments, the cells arehematopoietic cells, such as, for example, hematopoietic stem orprogenitor cells (e.g., isolated from umbilical cord blood or mobilizedperipheral blood), optionally treated with one or more agents toincrease one or more therapeutic properties of the cells. In a certainembodiment, the cells are treated with a prostaglandin pathway agonist,e.g., 16,16-dmPGE2, optionally at a concentration of 10 for a time ofabout 2 hours, at 37° C.

Administration of an “amount” of cells prepared herein to a subjectrefers to administration of “an amount effective,” to achieve thedesired therapeutic or prophylactic result, including withoutlimitation, treatment of the subject. A “therapeutically effectiveamount” of cells for purposes herein is thus determined by suchconsiderations as are known in the art, and may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the cells to elicit a desired response inthe individual. The term “therapeutically effective amount” includes anamount that is effective to “treat” a subject (e.g., a patient). Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the cells are outweighed by the therapeuticallybeneficial effects.

A “prophylactically effective amount” refers to an amount of cellshaving therapeutic potential that is effective to achieve the desiredprophylactic result. Typically but not necessarily, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount is less than the therapeuticallyeffective amount.

Suitable methods for administering populations of cells used in themethods described herein include parenteral administration, including,but not limited to methods of intravascular administration, such asintravenous and intraarterial administration. Additional illustrativemethods for administering cells of the invention include intramuscular,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

In various embodiments, the blood cell product or hematopoietic cellsadministered to a subject are a heterogeneous population of cellsincluding, whole bone marrow, umbilical cord blood, mobilized peripheralblood, hematopoietic stem cells, hematopoietic progenitor cells, and theprogeny of hematopoietic stem and progenitor cells, includinggranulocytes (e.g., promyelocytes, myelocytes, metamyelocytes,neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes,erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producingmegakaryocytes, platelets), and monocytes (e.g., monocytes,macrophages).

Particular embodiments of the present invention now will be describedmore fully by the following examples. This invention may, however, beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will be thorough and complete, and willfully convey the scope of the invention to those skilled in the art.

EXAMPLES Example 1 STEMSPAN-ACF+Dextran-40 Minimizes Loss of ViableCD34+ Cells and Improves TNC Recovery During Short-Term Incubation ofWhole Cord Blood CD34 Enumeration of Cord Blood During Short-TermIncubation

Cord blood units were obtained from the Carolinas Cord Blood Bank (CBB).Samples from these cord blood units were thawed in STEMSPAN-ACF (StemCell Technologies, Vancouver, BC, Canada) or STEMSPAN-ACF with 8%Dextran-40 (Sigma-Aldrich, St Louis, Mo.) at 37° C. Each sample wascentrifuged at 400×g for 10 minutes and resuspended in the same mediumin which each sample was initially thawed. Both samples were incubatedfor 2 hours at 37° C. 100 uL samples were drawn from both conditionsthroughout the processing and incubation. BD Stem Cell Enumeration Kit(BD Biosciences, San Jose, Calif.) standard no-lyse, no-wash protocolfor cord blood units was used to stain the cells for CD34-PE, CD45-FITC,and 7AAD. The samples were analyzed on a BD FACSCanto II (BDBiosciences, San Jose, Calif.) and gated using ISHAGE gating strategy.The percentage of granulocytes was estimated using a gate set around thegranulocyte population with characteristic high side scatter and lowCD45 surface expression.

Results

More viable CD34⁺ cells were enumerated in the samples incubated inSTEMSPAN-ACF with 8% Dextran-40 compared to STEMSPAN-ACF alone at onehour and after two hours of incubation (post-incubation) at 37° C. (FIG.1a ). Post-incubation, the number of viable CD34⁺ cells was 36% less insamples incubated in STEMSPAN-ACF compared to samples incubated inSTEMSPAN with Dextran-40.

The percentage of intact granulocytes in the CD45⁺ cell fractionincreased about 20% in the sample incubated in STEMSPAN-ACF with 8%Dextran versus STEMSPAN alone at one hour and after two hours ofincubation (post-incubation) at 37° C. (FIG. 1b ). This result indicatedthat a smaller fraction of granulocytes lysed during the incubation.

Example 2 STEMSPAN-ACF+Dextran-40 Decreases Loss of TNC in Whole CordBlood Treated with dmPGE₂

Ex Vivo Modulation of a Whole Cord Blood Unit with dmPGE₂

Cord blood units were obtained from Carolinas CBB and stored in liquidnitrogen (LN₂) vapor phase. The cord blood units were RBC-reduced andplasma-reduced prior to cryopreservation and stored in a volume of 25mL. The cord blood units were thawed in a 37° C. water bath and thevolume was brought to 45 mL in STEMSPAN-ACF, STEMSPAN-ACF+2.1% HSA,STEMSPAN+4.2% HSA, IMDM +4.2% HSA, or STEMSPAN-ACF with 8% Dextran-40. ASepax 2 cell processing system (Biosafe, Geneva, Switzerland) was usedto wash the cord blood units with the same media in which the cord bloodunits were initially thawed. The total volume of the washed cord bloodunits was 95 mL. dmPGE₂ was added to the cord blood units to a finalconcentration of 10 μM. The cord blood units were placed into aPlasmatherm device (Barkey, Leopoldshoehe, Germany) for 2 hours at 37°C. with constant paddle-mixing. The total nucleated cell (TNC) count wasdetermined using a Sysmex KX-21N (Sysmex America, Inc, Lincolnshire,Ill.) after thawing, washing, and incubation steps.

Results

The TNC count for the cord blood units processed in STEMSPAN-ACF or IMDMresulted in an average TNC loss of 65.4% from thaw to post-incubation ascompared to an 8.0% loss of TNC in STEMSPAN-ACF with 8% Dextran-40 (FIG.2). One observable difference between these units was the cellulardebris clumping in media without Dextran-40. Most of the loss of TNCoccurred during the incubation but some loss also occurred during thewashing of the cord blood unit.

Example 3 Whole Cord Blood Treated With dmpge₂ Maintains BiologicalActivity in STEMSPAN-ACF+Dextran-40 Addition of Dextran-40 toSTEMSPAN-ACF Does Not Inhibit Biological Activities

Cord blood mononuclear cells (All Cells, Emeryville, Calif.) were thawedin a 37° C. water bath and immediately diluted with 10 mL of pre-warmedIMDM with 10% fetal calf serum and 5 uL of DNase (Life Technologies,Grand Island, N.Y.). The cells were split three ways and centrifuged at300×g for 10 minutes. The cells were resuspended in STEMSPAN-ACF (Media1), STEMSPAN-ACF with 5% Dextran-40 (Media 2) or STEMSPAN-ACF with 10%Dextran-40 (Media 3). A subset, 1×10⁶ cells, from Media 1 were kept at4° C. and immediately stained with Lineage Mix-FITC, CXCR4-PE, CD34-APC,and CD45-V450 antibodies from BD Biosciences. All 3 media conditionswere split into a vehicle-treated cohort and a 10 μM dmPGE2-treatedcohort and incubated at 37° C. for 2 hours. The cells were washed withthe same media and incubated at 37° C. for an additional 1 hour. Eachcohort was stained with the same antibodies as indicated above. Levelsof CXCR4 surface protein within the Lin-CD45^(low)CD34+7AAD-cells wereanalyzed.

Results

After incubation with 10 μM dmPGE₂ for two hours at 37° C., increasedCXCR4 gene expression was observed, and after an additional one hourincubation without dmPGE2, CXCR4 surface protein was increased. Theaddition of 5% or 10% Dextran-40 to STEMSPAN-ACF did not decrease theresponse of CD34⁺ cells to dmPGE₂ as evidenced by the increased level ofCXCR4 surface protein expression (FIG. 3).

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1. A composition comprising: (a) about 1% to about 20% polysaccharide;(b) a chemically defined cell culture medium; (c) transferrin or atransferrin substitute and/or insulin or an insulin substitute; and (d)one or more antioxidants wherein said composition does not contain fetalcalf serum.
 2. The composition of claim 1, wherein the polysaccharide isa dextran.
 3. The composition of claim 1, wherein the polysaccharide isa dextran selected from the group consisting of: dextran-1, dextran-10,dextran-20, dextran-30, and dextran-40.
 4. The composition of claim 1,wherein the polysaccharide is dextran-40.
 5. The composition of claim 1,wherein the polysaccharide is a hydroxyethyl starch (HES).
 6. Thecomposition of claim 1, wherein the polysaccharide is a HES selectedfrom the group consisting of: hetastarch, hexastarch, pentastarch, andtetrastarch.
 7. The composition of claim 1, wherein the medium comprisesabout 1% to about 10% polysaccharide.
 8. The composition of claim 1,wherein the medium comprises about 1% to about 5% HSA.
 9. Thecomposition of claim 1, wherein the chemically defined cell culturemedium is selected from the group consisting of: Iscove's modifiedDulbecco's medium (IMDM), Dulbecco's modified Eagle medium (DMEM),Roswell Park Memorial Institute medium (RPMI) 1640 medium, McCoy's 5 Amedium, minimum essential medium alpha medium (alpha-MEM), basal mediumEagle (BME), Fischer's medium, medium199, and F-12K nutrient mixturemedium (Kaighn's modification, F-12K).
 10. The composition of claim 1,further comprising one or more growth factors or cytokines.
 11. Thecomposition of claim 1, wherein the chemically defined culture mediumcomprises one or more growth factors or cytokines selected from thegroup consisting of: flt3-ligand (FLT3); thrombopoietin (TPO), stem cellfactor (SCF), epidermal growth factor (EGF), transforming growthfactor-beta (TGF-β), basic fibroblast growth factor (bFGF),interleukin-3 (IL3), interleukin-6 (IL6), and interleukin-9 (IL9). 12.The composition of claim 1, further comprising an agent selected fromthe group consisting of a cAMP analogue or enhancer, a Gα-s activator,and a prostaglandin pathway agonist.
 13. The composition of claim 12,wherein the prostaglandin pathway agonist: (a) selectively binds thePGE2 EP2 or PGE2 EP4 receptor; (b) comprises PGE2, or a PGE2 analogue orderivative; (c) is selected from the group consisting of: PGE2,16,16-dmPGE2, 15(S)-15-methyl PGE2, 20-ethyl PGE2, and8-iso-16-cyclohexyl-tetranor PGE2; or (d) comprises 16,16-dmPGE2.
 14. Amethod of preparing a cell population comprising contacting the cellpopulation with the culture media according to claim
 1. 15. The methodof claim 14, wherein the cell population comprises a hematopoietic cellpopulation.
 16. The method of claim 15, wherein the hematopoietic cellpopulation comprises at least one of: hematopoietic stem cells (HSCs),hematopoietic progenitor cells (HPCs), and CD34+ cells.
 17. The methodof claim 16, wherein the hematopoietic cell population is a purifiedpopulation of CD34+ cells.
 18. The method of claim 14, wherein the cellpopulation comprises cyropreserved cells and contacting comprisesthawing the cryopreserved cells in the culture media.
 19. The method ofclaim 18, wherein the cell population comprises cryopreserved cells thathave been thawed prior to contacting the population of cells with theculture media.
 20. The method of claim 14, wherein cell lysis of thecontacted cell population is decreased about 10%, about 20%, about 30%,about 40%, or about 50%, compared to the cell lysis of a control cellpopulation that has been contacted with a control culture media.
 21. Themethod of claim 14, wherein cell lysis of the contacted cell populationis decreased about two-fold, three-fold, or five-fold compared to thecell lysis of a control cell population that has been contacted with acontrol culture media.
 22. The method of claim 14, wherein the recoveryof TNC from the contacted hematopoietic cell population is increasedabout two-fold, about three-fold or about five-fold compared to therecovery of TNC from a control hematopoietic cell population that hasbeen contacted with a control culture media.
 23. The method of claim 22,wherein the viability of the contacted CD34+ cells is increased by atleast about 10%, about 20%, about 30%, about 40%, or about 50% comparedto the CD34+ cell viability of a control cell population that has beencontacted with a control culture media.
 24. The method of claim 14,wherein the cell population is modulated ex vivo.
 25. The method ofclaim 24, wherein the modulation comprises contacting the cellpopulation with at least one agent selected from the group consistingof: a cAMP analogue or enhancer, a Ga-s activator, and a prostaglandinpathway agonist.
 26. The method of claim 25, wherein the prostaglandinpathway agonist: (a) selectively binds the PGE2 EP2 or PGE2 EP4receptor; (b) comprises PGE2, or a PGE2 analogue or derivative; (c) isselected from the group consisting of: PGE2, 16,16-dmPGE2,15(S)-15-methyl PGE2, 20-ethyl PGE2, and 8-iso-16-cyclohexyl-tetranorPGE2; or (d) comprises 16,16-dmPGE2.
 27. The method of claim 14, whereinthe cell population is contacted with the at least one agent for a timeof about one hour to about four hours.
 28. The method of claim 25,wherein the cell population is contacted with the at least one agent ata temperature of about 25° C. to about 37° C.
 29. The method of claim24, wherein engraftment, reconstitution, homing and/or proliferation ofthe cell population is increased in vivo, compared to a non-modulatedcell population.
 30. The method of claim 24, wherein expression of atleast two genes selected from the group consisting of: CREM, GEM, NR4A2,NR4A3, ILIA, COX2, HEY1, CXCL2, CXCL3, and ULBP2 is increased by abouttwenty-fold in the cell population compared to expression of the atleast two genes in a control non-modulated cell population.